Production of Nucleic Acid

ABSTRACT

A method for the production of nucleic acid encoding a target protein. The method comprises (a) providing an array of RNA or DNA molecules including one or more encoding the target protein; (b) generating a target protein from the array to form RNA-protein or DNA-protein complexes in which the RNA or DNA molecule is non-covalently or covalently bound to the complex; (c) separating the complexes into compartments wherein most or all of the compartments contain no more than one complex; (d) subjecting the complexes to reaction conditions which allow target protein activity; and (e) selecting nucleic acid encoding the target protein on the basis of the activity associated therewith, wherein when the complex is a DNA-protein complex in which the DNA is non-covalently bound, step b) is performed in the absence of separate compartments for each complex.

This application is a Division of U.S. patent application Ser. No.15/584,565, filed May 2, 2017, which is a divisional of U.S. patentapplication Ser. No. 13/921,989, filed Jun. 19, 2013, now U.S. Pat. No.9,683,251, which is a Division of U.S. application patent applicationSer. No. 13/408,732 filed Feb. 29, 2012, now U.S. Pat. No. 8,580,548;which is a Continuation of U.S. patent application Ser. No. 12/899,641filed on Oct. 7, 2010, now U.S. Pat. No. 8,835,148; andPCT/EP2009/054329 filed Apr. 9, 2009; which claims priority to GreatBritian Application Serial No. 0806562.5 filed Apr. 10, 2008 each ofwhich is expressly incorporated by reference herein in its entirety.

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronicformat. The Sequence Listing is provided as a file entitled“2018-06-27_01129-0016-04US_Replacement_Seq_List.txt” created on Jun.27, 2018, which is 667,739 bytes in size.

FIELD OF INVENTION

A method for the production of nucleic acid encoding a target proteinand target protein obtainable thereby, including enzymes such as nucleicacid processing enzymes, e.g., reverse transcriptase.

BACKGROUND OF THE INVENTION

Protein evolution is a known technology for selection and directedevolution of proteins from large libraries. The basic principle ofselection is to ensure that there is a linkage between a specificphenotype (protein) and its encoding genotype. This phenotype-genotypelinkage can be realized in three different ways:

-   -   covalent linkage such as mRNA display, and to some extent phage        display, bacterial display, yeast display etc.,    -   non-covalent linkage which use affinity interaction. Examples        are ribosome display, CIS display, plasmid display etc.,    -   compartmentalization such as in vitro compartmentalisation        (IVC), compartmentalized self-replication (CSR), simple        bacterial screening, high throughput screening etc.

As indicated above, one example of covalent phenotype-genotype linkageis achieved using mRNA display. As described by Roberts and Szostak(1997) covalent fusions between an mRNA and the peptide or protein thatit encodes can be generated by in vitro translation of synthetic mRNAsthat carry puromycin, a peptidyl acceptor antibiotic, at their 3′ end.

Non-covalent linkage between phenotype and genotype can be achieved withribosome display. In ribosome display, an array of RNAs including one ormore encoding a target protein is subjected to an in vitro translationsystem so as to form a non-covalent ternary complex of ribosome, mRNAand protein coupled to tRNA. This array of ternary complexes must bestabilized. Accordingly, each ternary complex formed during in vitrotranslation uses mRNA lacking a STOP codon at the end. The ternarycomplexes are further stabilized at low temperature (4° C.) and highconcentration of magnesium (50 mM). In the stable complex the linkagebetween phenotype and genotype is preserved. A selection step followswhereby target protein is selected on the basis of a property of theprotein whilst still attached to the ternary complex. Selected ternarycomplexes may then be disassembled and the mRNA associated with thetarget protein is amplified by RT-PCR.

In general, ribosome display is successfully applied for the selectionof peptides (Mattheakis et al., 1994; Matsuura and Pluckthun, 2003) andproteins (Hanes and Pluckthun, 1997; He and Taussig, 1997; Irving etal., 2001), which bind to different targets. In some cases it ispossible to use ribosome display to select for enzymatic activities,performing affinity selection of proteins with suicide inhibitor(Amstutz et al., 2002) or active site ligand (Takahashi et al., 2002).

In in vitro compartmentalization (IVC) phenotype and genotype linkage isrealized by in vitro compartmentalization of genes in a water-in-oilemulsion. The number of genes used to prepare the emulsion is calculatedsuch that most of water compartments contain no more than a single gene.The compartmentalized genes are transcribed and translated. The activityof the synthesized proteins is then assessed. Subsequent selection onthe basis of protein activity results in amplification of DNA encodingactive proteins with desired properties. Water droplets used in most IVCapplications are 2-3 μm in size, giving ˜5 femtoliters reaction volumeand ˜10¹⁰ water-in-oil compartments (50 μl water phase) per 1 ml ofemulsion. The first successful example of IVC selection system was basedon target-specific DNA methylation activity (Tawfik and Griffiths,1998). Genes of HaeIII methyltransferase were compartmentalized,transcribed and translated. In vitro synthesized methyltransferase inthe presence of a cofactor was able to methylate its own DNA. Based onmethylated DNA (reaction product) resistance to digestion with HaeIIIrestriction endonuclease, genes encoding methyltransferase were selectedfrom a 10⁷ fold excess of other DNA molecules.

To date many more modifications of IVC have been designed and realized.The easiest way to perform IVC selection is to use DNA-modifyingenzymes, in particular DNA-methyltransferases (Lee et al., 2002; Cohenet al., 2004). A similar experimental strategy was applied in order toselect for active variants of FokI restriction endonuclease (Doi et al.,2004). Compartmentalized DNA, encoding active restriction endonucleases,was digested and selected by subsequent incorporation of biotin-dUTP andbinding to streptavidin beads.

A different IVC selection strategy was applied for evolution of DNApolymerases (Ghadessy et al., 2001; Ghadessy et al., 2004; Ong et al.,2006). This new selection method is based on ‘compartmentalizedself-replication’ (CSR) of genes encoding active DNA polymerase.Contrary to usual IVC, where proteins of interest are expressed in situ,CSR is performed by compartmentalization of bacterial cells expressingthermophilic DNA polymerase. Cells resuspended in PCR buffer,supplemented with primers and dNTP's, are emulsified yieldingcompartments ˜15 μm in size. Each water droplet serves as a separate PCRcompartment. During the initial PCR denaturation step the bacterialcells are broken releasing the expressed thermophilic DNA polymerase andits encoding gene into the reaction mixture, allowing self-replicationto proceed meanwhile other bacterial proteins are denatured by hightemperature.

A modification of IVC is the use of double water-in-oil-in-wateremulsions. Water droplets surrounded by an oil layer can be analyzed andsorted in a FACS at a rate of >10⁴ variants per second (Bernath et al.,2004; Mastrobattista et al., 2005).

Selection of proteins for binding may also be performed by IVC. Aprotein of interest expressed in water-in-oil compartments is coupledcovalently (Bertschinger and Neri, 2004) or non-covalently (Doi andYanagawa, 1999; Yonezawa et al., 2003; Sepp and Choo, 2005) to the genethat encodes it.

Other applications of IVC are known where microbeads entrapped intocompartments are used as intermediators for protein and gene coupling.Single genes attached to microbeads are transcribed and translated inwater droplets. Newly synthesized protein is captured onto the same beadwithin reaction compartment. After emulsion is broken isolated beads canbe used further for affinity selection (Sepp et al., 2002). Emulsionsusually are broken using organic solvents (hexane, ether, chloroform),which can also decrease activity of certain enzymes displayed on thebeads, limiting the application of this technology. For selection ofcatalytic activity microbeads can be easily washed, resuspended indifferent reaction buffer and compartmentalized again by secondemulsification step (Griffiths and Tawfik, 2003). However sometimesrigid enzyme-bead-gene complexes (because of steric hindrance and enzymemobility limitations) cannot fulfill essential reaction requirements andthe activity of the attached enzymes may be lower than that of the freeenzyme. In addition, such methods are technically complicated since manyadditional components have to be used (i.e. affinity tags, antibodies,and beads).

The composition of emulsions used in IVC is designed to ensure stabilityof water compartments and efficient in vitro transcription of mRNA andsubsequent translation of target proteins. In vitro evolution has abroad range of targets to be improved. Some proteins and enzymes ofinterest are robust hard-workers and can be active enough in differentbuffers, in particular in reaction mixtures used in IVC. However thereare many complicated enzymes, which can work only under optimized orspecific conditions. In addition to that the first law of in vitroevolution says “you will evolve what you are selecting for”. That means,an enzyme evolved and optimized in transcription/translation reactionmixture will work well in that particular mixture and most likely willperform much worse in its own buffer. In some cases enzyme workingconditions are incompatible with in vitro transcription and translationmixture used for protein expression in compartments. A partial solutionis a nano-droplets delivery system (Bernath et al., 2005) used totransport different solutes into emulsion compartments. Even moresophisticated manipulations with water-in-oil compartments can be doneemploying microfluidics devices. Highly monodisperse single or doubleemulsions (Thorsen et al., 2001; Okushima et al., 2004) can be preparedat a rate of up to 10000 aqueous droplets per second. Generated watercompartments can be transported in microfluidic channels, fused,subdivided and sorted (Song et al., 2003; Link et al., 2006).Nevertheless full buffer exchange in the compartments is still aproblem.

Reverse transcriptases are very important commercial enzymes used tosynthesize cDNA from an mRNA target. A lot of research has been done inorder to improve properties of reverse transcriptases. However noproperly working selection system suitable for in vitro evolution ofreverse transcriptase was known to date. Almost all improvements aremade and mutants of reverse transcriptase selected using high throughputscreening and rational design.

Neither ribosome display (RD), nor in vitro compartmentalization (IVC)may be used for selection of fully active reverse transcriptase. Ternarycomplexes used in ribosome display usually are not stable at highertemperatures required to perform reverse transcriptase selection and asa consequence linkage between phenotype and genotype will be lost. Whilerelatively stable ternary complexes used in ribosome display can beproduced using synthetic in vitro translation extract WakoPURE (Matsuuraet al., 2007), in vitro translated reverse transcriptase immobilized toribosome and mRNA will encounter significant steric hindrance duringsynthesis of full-length cDNA. There is also the possibility thatimmobilized enzyme will act in trans as well as in cis, which again isincompatible with protein evolution strategy because thephenotype-genotype linkage will not be preserved.

IVC generally employs DNA as genetic material. In vitro transcription ofmRNA and target protein translation is performed in spatially separatedemulsified water compartments in the presence of the DNA. In the case ofreverse transcriptase in vitro evolution, the presence of coding DNAsequence abolishes the main prerequisite of selection for reversetranscriptase activity—cDNA has to be synthesized de novo. In otherwords, selection for better reverse transcriptase variants is based onenzyme ability to synthesize their own coding cDNA from mRNA. Newlysynthesized cDNA has to be amplified by PCR, therefore cDNA should bethe only source of DNA in the reaction. DNA used in IVC selection willamplify together with cDNA canceling basic selection scheme.

More sophisticated variants of in vitro compartmentalization, such asuse of microbeads entrapped in water compartments (Sepp et al., 2002;Griffiths and Tawfik, 2003), allow complete exchange of reaction buffer.In this approach the phenotype-genotype linkage is realized viamicrobeads yielding a rigid selection unit mRNA-microbead-protein which,in case of reverse transcriptase selection, again can cause sterichindrance and as a result inefficient cDNA synthesis.

Taq DNA polymerase able to synthesize ˜300 nucleotides long cDNA wasselected using modification of phage display technology (Vichier-Guerreet. al, 2006). Although this approach works, it has someshortcomings: 1) not all proteins can be displayed on phages; 2)absolute requirement to use biotin labeled nucleotides for selection; 3)displayed enzyme can work in trans as well as in cis; 4) because ofsteric hindrance and enzyme mobility limitations phage-enzyme-DNA/RNAcomplex can interfere with efficient synthesis of cDNA.

The possibility of selection for reverse transcriptase is also mentionedin WO 0222869, which is related to the compartmentalizedself-replication (CSR) method. CSR technology is used to selectthermophilic DNA polymerases, in particular Taq DNA polymerase (Ghadessyet al., 2001; Ghadessy et al., 2004; Ong et al., 2006). Bacterial cells,expressing mutants library of thermophilic DNA polymerase are suspendedin PCR mixture and emulsified yielding separate PCR compartments for invitro selection of more active polymerases.

Real selection for reverse transcriptase activity will be prevented bythe presence of bacterial RNases, which remain active at moderatetemperatures and will degrade target mRNA. There will also be DNAcontamination from non-selected plasmid DNA (released from bacterialcell) as well as the presence of all E. coli enzymes, structuralproteins, ribosomes, NTP, RNases, DNases and small molecular weightmolecules.

SUMMARY OF THE INVENTION

The invention aims to provide an improved method of protein evolutionwhich does not suffer from drawbacks of the prior art methods.

Accordingly, in a first aspect, the present invention provides a processfor the production of nucleic acid encoding a target protein, whichcomprises:

(a) providing an array of RNA or DNA molecules including one or moreencoding the target protein;

(b) generating a target protein from the array to form RNA-protein orDNA-protein complexes in which the RNA or DNA molecule is non-covalentlyor covalently bound to the complex;

(c) separating the complexes into compartments wherein most or all ofthe compartments contain no more than one complex;

(d) subjecting the complexes to reaction conditions which allow targetprotein activity; and

(e) selecting nucleic acid encoding the target protein on the basis ofthe activity associated therewith, wherein when the complex is aDNA-protein complex in which the DNA is non-covalently bound, step b) isperformed in the absence of separate compartments for each complex.

The method uses two distinct types of phenotype-genotype linkage andachieves a new selection system for use in methods of protein evolutionwithin the laboratory. The new features of the method, described furtherbelow, allow it to be applied to an extended range of target proteins incomparison to the methods of the prior art, with increased ease of useand flexibility. In particular, the present invention provides for thefirst time the possibility of evolving and improving the properties ofreverse transcriptase enzymes, one of the most important enzyme groupsin the toolbox of molecular biologists.

In one embodiment of this aspect the invention provides a process forthe production of nucleic acid encoding a target protein, whichcomprises:

(a) providing an array of RNA or DNA molecules including one or moreencoding the target protein;

(b) generating a target protein from the array to form RNA-protein orDNA-protein complexes;

(c) separating the complexes into compartments wherein most or all ofthe compartments contain no more than one complex;

(d) subjecting the complexes to reaction conditions which allow targetprotein activity; and

(e) selecting nucleic acid encoding the target protein on the basis ofthe activity associated therewith, wherein in the RNA-protein complexthe RNA is non-covalently or covalently bound thereto and in theDNA-protein complex the DNA is covalently bound thereto.

In a second embodiment of this aspect the invention provides process forthe production of nucleic acid encoding a target protein, whichcomprises:

(a) providing an array of RNA or DNA molecules including one or moreencoding the target protein;

(b) generating a target protein from the array to form RNA-protein orDNA-protein complexes in which the RNA or DNA molecule is non-covalentlyor covalently bound to the complex;

(c) separating the complexes into compartments wherein most or all ofthe compartments contain no more than one complex;

(d) subjecting the complexes to reaction conditions which allow targetprotein activity; and

(e) selecting nucleic acid encoding the target protein on the basis ofthe activity associated therewith, wherein step b) is performed in theabsence of separate compartments for each complex.

Covalent linkages between DNA or RNA and the target protein can begenerated by any technique known in the art, for example, mRNA display,phage display, bacterial display or yeast display. In particular, acovalent RNA-protein linkage can be generated using the technique ofmRNA display, while a covalent DNA-protein linkage can be generatedusing the technique of covalent antibody display (CAD) (Reiersen et al.,2005), performing translation in compartments by covalent DNA display(Bertschinger and Neri, 2004), or using similar covalent displaytechniques such as those described by Stein et al., (2005).

Non-covalent linkages between DNA or RNA and the target protein can alsobe generated by any technique known in the art, e.g., ribosome display,CIS display, or plasmid display. In particular, a non-covalentDNA-protein linkage can be generated in the absence of a compartmentusing CIS display (Odergrip et al., 2004), while a non-covalentRNA-protein linkage can be generated using the technique of ribosomedisplay.

As indicated above, when the complex is a DNA-protein complex in whichthe DNA is non-covalently bound, step (b) of the process of theinvention is performed in the absence of separate compartments for eachcomplex. In other words, step (b) is un-compartmentalized. Specifically,when the complex generated is a DNA-protein complex in which the DNA isnon-covalently bound, the generation step is performed withoutseparating each member of the array from one another. In particular, thegeneration step is performed without separating each member of the arrayby in vitro compartmentalization (IVC). In a particularly preferredembodiment the generation step is performed without separating eachmember of the array in a water-in-oil emulsion.

Compartmentalization can also be performed by any method known in theart that enables the complexes to be generated or separated such thatall or substantially all of the compartments contain no more than onecomplex. In particular, it is preferred that at least 70%, at least 80%or at least 90% of the compartments contain no more than one complex.For example, compartmentalization can be performed by separating membersof the array or each complex into different wells on a microliter ornanowire plate, or by in vitro compartmentalization (IVC). Inparticularly, separation by IVC can involve separation into aqueousdroplets in a water-in-oil emulsion or a water-in-oil-in-water emulsion.

The disclosed method combines at least two different types ofgenotype-phenotype linkage selected from covalent linkage, non-covalentlinkage, and compartmentalization. In a preferred aspect of theinvention the method utilized no more than two of these linkages. Thusin a particularly preferred embodiment the method utilizes the covalentor non-covalent linkage as the only genotype-phenotype linkage in stepb). In other words, in this embodiment there is no compartmentalizationin step b).

Covalent/non-covalent linkages between DNA or RNA and the protein in theabsence of a compartment can be established in many different ways,e.g., by ribosome display, mRNA display (Roberts and Szostak, 1997), CISdisplay (Odergrip et al., 2004), or covalent antibody display (CAD)(Reiersen et al., 2005). Specifically, covalent DNA-protein linkage canbe realized in the absence of compartments by using CAD technique, whilecovalent RNA-protein linkage and non-covalent RNA-protein linkage can beestablished by mRNA display and ribosome display, respectively.

The disclosed invention can be realized through a combination of manydifferent linkages. For example, the present invention can be realizedthrough a combination of ribosome display and in vitrocompartmentalization, or through a combination of mRNA display, CISdisplay, or CAD display and IVC.

In a preferred aspect the process for the production of nucleic acidencoding a target protein is realized through a combination of ribosomedisplay and in vitro compartmentalization. In particular such an processcomprises:

(a) providing an array of mRNAs including one or more encoding thetarget protein;

(b) incubating the array of mRNAs under conditions for ribosometranslation to generate an array of ternary complexes each comprising anmRNA, a ribosome and protein translated from the mRNA;

(c) incorporating the array of ternary complexes into aqueous phasedroplets of a water-in-oil or a water-in-oil-in-water emulsion, wheremost or all of the aqueous phase droplets contain no more than oneternary complex;

(d) subjecting the aqueous phase droplets to reaction conditions whichallow protein activity; and

(e) selecting nucleic acid encoding the target protein on the basis ofthe enzyme activity associated therewith.

Such a process may be termed “compartmentalized ribosome display” (CRD).CRD is applicable to a wide range of target proteins, including enzymes.CRD has the advantage that the linkage between the enzyme and the mRNAis non-covalent. Thus if the reaction conditions used in step (d)involve a raised temperature the ternary complexes generated in step (b)will fall apart and the enzyme will be released. This avoids theproblems associated with enzyme mobility described above for prior artmethods in which the enzyme is immobilized on a bead.

Emulsion droplets with ribosome display complexes inside can be sortedor selected in step (e) in many ways. Preferably they are sorted byfluorescence activated cell sorting (FACS) or using microfluidictechniques. Both techniques mainly exploit fluorescence based dropletsorting. However, droplets can also be separated by size, lightdiffraction or light absorption, depending on the reaction conditionsused in step (d) and the protein activity that is being selected for.

Fluorescent based sorting methods are preferably used when the targetprotein is an enzyme. In this embodiment the reaction conditionsemployed in step (d) include a non-fluorescent substrate capable ofbeing converted to a fluorescent product. Activity by the enzymegenerates the fluorescent product, allowing FACs to be used todistinguish between fluorescent droplets containing an active enzyme andnon- or less fluorescent droplets which contain no active enzyme or aless active enzyme.

In particular, CRD is applicable to nucleic acid processing enzymes suchas reverse transcriptases, and allows for fast and efficient in vitroevolution.

In a further aspect the invention provides a process for the productionof nucleic acid encoding a target protein, which comprises:

(a) providing an array of mRNAs including one or more encoding thetarget protein, wherein the mRNAs comprise a substrate for an enzymecomprising the target protein or a co-enzyme thereof;

(b) incubating the array of mRNAs under conditions for ribosometranslation to generate an array of ternary complexes each comprising anmRNA, a ribosome and protein translated from the mRNA;

(c) incorporating the array of ternary complexes, and optionally theco-enzyme, into aqueous phase droplets of a water-in-oil or awater-in-oil-in-water emulsion, wherein most or all of the aqueous phasedroplets contain no more than one ternary complex;

(d) subjecting the aqueous phase droplets to reaction conditions whichallow enzyme activity; and

(e) selecting nucleic acid encoding the target protein on the basis ofthe enzyme activity associated therewith.

In one embodiment of this aspect of the invention, where the nucleicacid processing enzyme is a DNA dependent DNA polymerase, the mRNA ofstep (a) can be ligated to a double stranded DNA adaptor molecule toprovide the substrate.

CRD diversity is ˜10⁹-10¹⁰ variants and is limited by IVC step. Theinventive method is much more efficient, less time consuming and cheapercompared to high throughput screening (HTS), which can be used to screen˜10⁵-10⁶ mutant variants of reverse transcriptase. CRD diversity isabout four orders of magnitude higher compared to HTS; thus many morebeneficial mutants missed by HTS can be easily fished-out bycompartmentalized ribosome display selection.

According to step (a), an array of mRNAs which are typically synthesizedmRNAs is provided which includes one or more members of the arrayencoding the target protein. Where the target protein is reversetranscriptase the mRNAs comprise a substrate for an enzyme comprisingthe target protein or a co-enzyme thereof. In the subsequent selectionstep (e), the nucleic acid encoding the target protein is selected onthe basis of the enzyme activity associated therewith. In this way, twoembodiments of the invention are contemplated: one in which the enzymeactivity is provided by the target protein and one in which the enzymeactivity is provided by a co-enzyme of the target protein in thepresence of the target protein. In the embodiment requiring theco-enzyme, this is incorporated into aqueous phase droplets of thewater-in-oil emulsion of step (c), as is the array of ternary complexes.Where the enzyme comprises the target protein, no additional co-enzymeneed to be incorporated into the aqueous phase droplets.

In step (b) of the process, the array of mRNAs is treated with ribosomesto generate an array of ternary complexes, each comprising an mRNA, aribosome and protein translated from the mRNA. This step may beperformed under any conditions suitable for typical in vitro translationof mRNA, as used for example in the technique of ribosome display. Atthis point, the ternary complexes may be purified, although this is notessential. The ternary complexes may be supplemented at this point withany co-substrates necessary for the subsequent enzyme activity whereuponthe reaction mixture is typically emulsified to give approximately 10¹⁰water-in-oil compartments each typically having a mean diameter ofapproximately 2 to 3 μm. Even a small volume (25 μl) of in vitrotranslation reaction generates approximately 10¹¹ to 10¹² molecules ofstored ribosomal complexes. A typical ribosome display method uses mRNAslacking STOP codons, although STOP codon may be present (Matsuura etal., 2007). In order to achieve aqueous phase droplets in which most orall contain no more than one ternary complex the concentration ofternary complexes would have to be reduced by about two orders ofmagnitude as compared with corresponding concentration used in a typicalribosome display technique. Only a very small concentration of ternarycomplexes is used in this step of the process.

The enzyme may comprise a nucleic acid processing enzyme, which may bean RNA processing enzyme. The nucleic acid processing enzyme maycomprise the target protein and may be selected from a nucleic acidpolymerase, a nucleic acid ligase, and a terminal deoxynucleotidyltransferase. As described in further detail herein, the nucleic acidpolymerase may comprise a reverse transcriptase. In this embodiment, themRNA encoding the reverse transcriptase is itself the substrate for thereverse transcriptase. Step (e) of selecting nucleic acid encoding thetarget protein, comprises selecting cDNA produced by the action of thereverse transcriptase, which cDNA encodes reverse transcriptase.

Where the target protein is a nucleic acid ligase, selection for RNA(DNA) ligases able to ligate RNA to RNA or DNA to RNA can be performed.Preferably, the reaction conditions which allow enzyme activity includea co-substrate comprising a nucleic acid linker or adaptor, whichco-substrate further comprises an affinity ligand for attachment to aligand binding partner or sequence tag for specific amplification ofprocessed mRNA in RT-PCR. In the first case, the mRNA encoding thetarget protein is ligated to the co-substrate in those aqueous phasedroplets which incorporate mRNA encoding a nucleic acid ligase.Preferably, the affinity ligand comprises biotin and the ligand bindingpartner comprises streptavidin. The step of selecting nucleic acidencoding the target protein comprises selecting mRNA incorporating theco-substrate by attachment to a solid phase comprising the ligandbinding partner. In a typical process, a mutant library of ligase istranslated in vitro and purified ternary complexes are diluted andemulsified in reaction buffer with biotin labeled DNA/RNA linker and/oradaptor. After bringing the emulsion to a temperature of 37° C. ribosometernary complexes disassemble. Ligase will be released and the 3′ end ofthe mRNA will become accessible for the biotin labeled adaptor andsubsequent ligation reaction. Biotin labeled mRNA encoding only active(or more active) variants of ligase will be purified on the streptavidinbeads and may be amplified by RT-PCR.

In the second case the step of selecting nucleic acid encoding thetarget protein comprises selecting mRNA with attached sequence specifictag, which can be used for selective annealing site of primer forreverse transcription and subsequent PCR.

In a typical process, a mutant library of ligase is translated in vitroand purified ternary complexes are diluted and emulsified in reactionbuffer with DNA/RNA linker and/or adaptor. After bringing the emulsionto a temperature of 37° C. ribosome ternary complexes disassemble.Ligase will be released and the 3′ end of the mRNA will becomeaccessible for the adaptor and subsequent ligation reaction. RNAencoding only active (or more active) variants of ligase will havespecific linker sequence required for specific annealing of primer usedin reverse transcription and may be efficiently amplified by RT-PCR.

It is also possible to select for terminal deoxynucleotidyl transferase(TdT). This enzyme works on RNA and incorporates deoxyribonucleotides,ribonucleotides, nucleotide analogues and similar. In this embodiment,the reaction conditions which allow enzyme activity include aco-substrate comprising dNTP which further comprises an affinity ligandfor attachment to a ligand binding partner. As with the nucleic acidligase, the affinity ligand may be biotin and the ligand binding partnerstreptavidin. Selecting nucleic acid encoding the target protein maycomprise selecting mRNA incorporating the co-substrate by attachment toa solid phase comprising the ligand binding partner. A mutant library ofTdTs may be translated in vitro and purified ribosome ternary complexesmay have to be diluted and emulsified in reaction buffer with biotinlabeled nucleotides, such as biotin-dUTP. The optimal workingtemperature for wild type enzyme is 37° C. At this temperature theribosome ternary complexes will disassemble and the 3′ end of mRNA willbecome accessible for template independent polymerization reaction.TdT-encoding mRNA incorporating biotin labeled nucleotide is selected onthe streptavidin beads and may subsequently be reversed transcribed andamplified by RT-PCR.

In a further embodiment, the target protein comprises a reversetranscriptase helper enzyme such as a helicase, pyrophosphatase,processivity factor, RNA binding protein or other protein able toimprove a reverse transcription reaction in the presence of reversetranscriptase. In this embodiment, the nucleic acid processing enzymecomprises a reverse transcriptase, which is the co-enzyme incorporatedinto the aqueous phase droplets of the water-in-oil emulsion. In step(e) of selecting nucleic acid target protein, cDNA is selected, whichcDNA is produced by the action of the reverse transcriptase and whichencodes the reverse transcriptase helper enzyme. The presence of thereverse transcriptase helper enzyme in the aqueous phase facilitatesreverse transcription of the mRNA which encodes the helper. Thus, mRNAwhich is reverse transcribed forms cDNA which encodes the helper andthis may be PCR amplified.

In a further embodiment, the target protein comprises an RNaseinhibitor. In this embodiment, the nucleic acid processing enzymecomprises an RNase. The step (e) of selecting nucleic acid encoding thetarget protein comprises selecting mRNA undegraded by RNase. In thisembodiment, the RNase is incorporated as the co-enzyme into the aqueousphase droplets of the water-in-oil emulsion. Once reaction conditionsallow enzyme activity, any droplets not containing effective RNaseinhibitor would exhibit RNase activity whereby the mRNA would bedegraded. Thus, mRNA encoding RNase inhibitor effective at the reactionconditions used would survive. Typically, a mutant library of RNaseinhibitors is translated in vitro and purified ribosome ternarycomplexes are diluted and emulsified in reaction buffer with appropriateRNase. In an alternative arrangement RNase can be delivered later byemulsion micro droplets. mRNA encoding only active (or more stable)RNase inhibitor will be purified and amplified by RT-PCR.

Compartmentalized ribosome display can also be used for reaction bufferexchange in in vitro compartmentalization where selection buffer isincompatible with in vitro translation mixture and substrate conversionto product has to be performed under strictly controlled reactionconditions.

The nucleic acid encoding the target protein which has been selected onthe basis of the enzyme activity associated therewith may be DNA or RNA,as discussed herein. The array may be converted or amplified to form DNAor RNA. In a preferred arrangement, the array is converted or amplifiedto form the array of mRNAs of step (a) of the process and is subject toone or more further cycles of steps (b) to (e) so as to enrich furtherthe array with increasing amounts of mRNAs encoding the target protein.

The step (d) of subjecting the aqueous phase droplets to reactionconditions which allow enzyme activity provides the basis for selectionstep (e) where those nucleic acids encoding the target protein areselected. A wide range of reaction conditions may be used in step (d) toprovide selection pressure. In one example, the reaction conditionsinclude a temperature above the optimum temperature for a wild typeenzyme. These reaction conditions may be used to select a mutant enzymewhich is more thermostable than wild type enzyme or which has a greaterreaction velocity at that temperature or an altered temperature-activityprofile. Mutant enzymes may have to operate at higher sensitivities thanwild type enzymes because concentrations of mRNA in the aqueous phasedroplets are approximately 400 pM. Mutant enzymes may also have toperform more accurately. All of these selection pressures areparticularly important in relation to reverse transcriptases. As well asphysical conditions, the reaction conditions may include alterations inbuffer, concentrations of other factors such as metal ions and pH.

Many more different selection pressures can be applied in CRD selectingfor better reverse transcriptases: 1) selection for more soluble enzymeswhich are less prone to aggregation—ternary complexes (beforeemulsification) have to be preincubated with hydrophobic material inorder to eliminate proteins with surface exposed hydrophobic residues;2) selection for very fast enzymes—reverse transcription reaction timeshave to be gradually reduced during selection cycles; 3) selection forenzymes synthesizing long cDNA—gradual prolongation of mRNA library usedin CRD and as a consequence synthesis of longer cDNA; 4) selection forenzymes able to transcribe through secondary structures—secondarystructure forming sequences have to be introduced into mRNA library usedin CRD; 5) selection for enzymes working in buffers which are differentfrom RT buffer (for example in PCR, one step RT-PCR buffer or withdenaturing agents)—CRD selection has to be performed in buffer of ourchoice; 6) selection for enzymes able to incorporate nucleotideanalogues—selection has to be performed in RT buffer with biotin labelednucleotide analogues with subsequent cDNA purification on streptavidinbeads.

Compartmentalized ribosome display (CRD) is also suitable for manyfluorescence activated cell sorting (FACS) applications. Protein ofinterest has to be displayed in ribosome display format. Optionallypurified (or just diluted many times) ternary complex comprisingmRNA-ribosome-protein (tRNA) mixed with non-fluorescent substrate (S) inreaction buffer should be emulsified producing doublewater-in-oil-in-water emulsions (Bernath et al., 2004; Mastrobattista etal., 2005). Active variants of compartmentalized enzymes will convertsubstrate (S) to fluorescent product (P) allowing FACS to distinguishbetween fluorescent (active enzyme inside) and “dark” (inactive enzymeinside) droplets. Contrary to previously published examples, whereenzymatic reaction has to be performed in transcription/translationmixture, CRD allows for complete buffer exchange and selection foractive enzymes in more native (required) conditions (FIG. 10.)

Selection and evolution of thermostable DNA polymerases using CRD isalso possible (FIG. 11.). Polymerase of interest has to be displayed inribosome display format. Optionally purified (or just diluted manytimes) ternary complex comprising mRNA-ribosome-polymerase can be usedto prepare reaction mixture with reverse transcriptase (helper enzyme),dNTP's and primer set in PCR buffer. Reaction solution should beemulsified producing water-in-oil emulsion. In first RT step—reversetranscriptase has to synthesize cDNA, which subsequently will serve as atarget for second PCR step—cDNA amplification by ribosome displayed DNApolymerase. One of the primers used in PCR can have biotin for optionalsubsequent purification with streptavidin beads and non-complementary 5′end. After RT-PCR emulsions should be broken, newly synthesized DNAfragment can be purified via biotin and reamplified using new primerset, which will contain one primer with sequence non-complementary tocDNA, but identical to 5′ part of primer used in first amplificationreaction (selective amplification of DNA over cDNA background). Moreactive variants of DNA polymerase will be enriched over less activevariants and can be used for further analysis or next selection round(FIG. 11.).

Features of the compartmentalized ribosome display technology are:

1) the genotype is maintained by an mRNA library, which is especiallyuseful in selecting RNA processing enzymes;2) the selection unit is a ternary complex of mRNA-ribosome-protein(tRNA) and this can readily purified by ultracentrifugation,gel-filtration, affinity tag purification and other simple means;3) the reaction buffer can be exchanged thereby enabling the selectionunit to be transferred (with or without purification) to a new reactionmixture and at the same time be diluted by a factor of 100 to 200 (inorder to adjust the number of ribosome complexes after emulsification toless than one molecule per reaction compartment);4) the mRNA library diversity of CRD is limited only by the diversity ofthe in vitro compartmentalization and is less than 10¹⁰ differentvariants;5) once emulsified, the selection reaction can be performed at a broadrange of temperatures from 4° to 94° C. because emulsions are stableover these temperatures;6) if selections performed at elevated temperatures (30° C. and above),the ternary complexes will dissociate but remain compartmentalized so asnot to lose the genotype-phenotype link, releasing mRNA and in vitrotranslated protein.

The inventive compartmentalized ribosome display process has been foundto work particularly well in promoting evolution of new reversetranscriptase enzymes. As an example, M-MuLV reverse transcriptase(Gerard et al., 1986, pRT601) may be used for selection (the enzymeincluding a N terminal His tag for purification). This reversetranscriptase has a temperature optimum for activity at 42° C. and isactive at temperatures up to 50° C. An array of mRNAs encoding theM-MuLV reverse transcriptase may be subjected in step (d) of the processof the present invention to reaction conditions which include primer anddNTPs required for cDNA synthesis at an incubation temperature in therange of from 50° C. to 60° C. At these elevated temperatures storedribosomal complexes stable at 4° C. quickly dissociate releasing intosolution a reverse transcriptase substrate (mRNA) and enzyme.

In one embodiment, in vitro translated reverse transcriptase M-MuLVreleased from ribosomal complex has C terminal fusion with phage lambdaouter surface protein D used in ribosome display construct as a spacerto remain in ribosomal tunnel (Matsuura and Pluckthun, 2003) andcovalently bound tRNA, because translation was not terminated properly.Protein D is very well expressed, soluble, stable protein with unfoldingtransition temperature ˜57° C. (Forrer and Jaussi, 1998) and thereforeis good fusion partner for selection of thermostable reversetranscriptase.

In compartmentalized ribosome display selection method only fully activevariants of reverse transcriptase can perform synthesis of cDNA, whichencodes the same active enzyme. In one hr, after the reversetranscription reaction is completed, emulsion is broken, cDNA ispurified and amplified by nested PCR. Because of the selection nature ofCRD only cDNA, which encodes active variants of reverse transcriptase,able to perform full length cDNA synthesis, will be amplified and ifnecessary transferred to the next selection round. In order to eliminateundesirable mutations in T7 polymerase promoter region, ribosome bindingsite (RBS) and protein D sequence, amplified DNA, encoding only M-MuLVsequence, may be ligated to native 5′ and 3′ terminal fragments in suchway that original ribosome display construct is restored and nextselection round can be performed.

In five selection rounds enzyme variants with specific activities at 50°C. have been identified, which are 2-4 times better as compared to theactivity of the primary enzyme used for library preparation. Some ofproteins are faster, some—more thermostable. Many selected M-MuLVvariants have mutations D524G or D583N, which turn off RNase H activityof reverse transcriptase and improve cDNA synthesis as well asthermostability (Gerard et al., 2002). Many more variants of selectedM-MuLV reverse transcriptases have other different beneficial mutations(H204R; H638R; T197A; M289V; E302K; T306A; N454K; Y64C; E69G; Q190R;V223M; F309S; L435P; E562K) mentioned and described before (U.S. Pat.No. 7,056,716; US20060094050A1; U.S. Pat. No. 7,078,208;US20050232934A1; WO07022045A2). In addition to that we have found manynew hot spots in reverse transcriptase amino acids sequence. Somemutations repeat very frequently and are of very high importance, whatwas shown analyzing purified mutants (Example 2). Thus the inventive CRDtechnology is very fast and robust selection method, efficiency of whichwas confirmed by direct evolution and improvement of M-MuLV reversetranscriptase. As proof of principle we selected variants of M-MuLVreverse transcriptase working better at higher temperatures.

In a further aspect, the present invention provides a reversetranscriptase enzyme obtainable by the process described herein.

In a further aspect, the present invention provides a reversetranscriptase enzyme having an optimum activity at a temperature above42° C., preferably at least 50° C. and more preferably in the range offrom 50° C. to 60° C. Reverse transcriptase enzymes may be selectedaccording to the process described herein by applying reactionconditions having an elevated temperature, preferably of at least 50° C.In this aspect of the invention, a reverse transcriptase enzyme may beselected which has an activity-temperature profile which is shifted incomparison with wild type enzyme to increase the temperature at whichoptimum activity is observed.

In a further aspect, the invention provides a reverse transcriptaseenzyme which comprises a MMLV reverse transcriptase amino acid sequencewith a mutation at one or more of the following amino acid positions:

D200, D653, L603, T330, L139, Q221, T287, I49, N479, H594, F625, H126,A502, E607, K658, P130, Q237, N249, A307, Y344, Q430, D449, A644, N649,L671, E673, M39, Q91, M66, W388, I179 E302 L333 R390 Q374 and E5

Where the mutation is at D653 it is preferred that the mutation is notD653N. Where the mutation is at L603 it is preferred that the mutationis not L603A. Further, where the mutation is at H594 it is preferredthat the mutation is not H594A.

It is preferred that the mutations at the above positions are pointmutations.

Preferably, the reverse transcriptase has one or more of the followingmutations:

D200N, A or, G, D653N, G, A, H or, V, L603W or M, T330P, L139P, Q221R,T287A, I49V or T, N479D, H594R or Q, F625S or L, H126S or R, A502V,E607K, G or A, K658R or Q P130S, Q237R, N249D, A307V, Y344H, Q430R,D449G or A, A644V or T, N649S, L671P, E673G or K, M39V or L, Q91R or L,M66L, W388R. I179T or V E302K L333Q R390W Q374R and E5K

Each of these mutations is found, e.g, in mutant enzymes having a higheractivity at 50° C. as compared with the corresponding wild type enzyme.Further details of these mutations are described in the specificexamples.

In particularly preferred aspect of the invention the mutant enzyme hasat least two mutations. In one embodiment the two mutations are at D200and at L603. For example the mutations are D200N and L603W. In analternative embodiment the mutations are at N479 and H 594. For examplethe mutations are N479D and H594R.

In a further aspect, the invention provides a reverse transcriptaseenzyme having an optimum activity at a temperature above 37° C., whereinthe activity at 50° C. is at least 120% of the activity at 37° C.Preferably, the activity at 50° C. is at least 130%, more preferably atleast 160% of the activity at 37° C.

In a further aspect, the present invention provides a mutant reversetranscriptase enzyme having an activity at 50° C. which is at leasttwice that of the corresponding wild type enzyme.

In a further aspect, the present invention provides a mutant reversetranscriptase enzyme having a specific activity at 37° C. which is atleast 130% of the corresponding wild type enzyme. Preferably, thespecific activity of the mutant reverse transcriptase enzyme is at least140%, more preferably at least 150% and particularly preferably at least160% of the specific activity of the corresponding wild type enzyme. Ithas been found as described herein that partially purified wild typeenzyme specific activity at 37° C. is approximately 200000μ/mg. Specificmutant reverse transcriptase enzymes obtainable in accordance with thepresent invention are discussed in further detail in the specificexamples.

In a further aspect, the present invention provides a mutant reversetranscriptase enzyme having a thermostability of at least 1.5 times thatof the corresponding wild type enzyme. Thermostability is measured inthe present application as residual activity at 37° C. followingtreatment at 50° C. for 5 minutes. Preferably, the thermostability ofthe mutant reverse transcriptase enzyme is at least 1.5 times, morepreferably at least 2 times, still more preferably at least 2.5 timesthat of the corresponding wild type enzyme. Typically, residual activityat 37° C. of the wild type reverse transcriptase enzyme is approximately11% as compared with untreated enzyme.

It is preferred that the reverse transcriptase enzyme according to theinvention comprises an MMLV reverse transcriptase.

In a further aspect, the present invention provides a polynucleotide,such as an mRNA or DNA, encoding a reverse transcriptase as describedherein.

The reverse transcriptases according to the present invention may beused in a variety of molecular biology techniques such as RT-PCR(qRT-PCR, etc). A kit for RT-PCR may be provided in which the reversetranscriptase of the kit is a reverse transcriptase according to thepresent invention.

DETAILED DESCRIPTION

The invention will now be described in further detail, by way of exampleonly, with reference to the accompanying figures and appendices:

FIG. 1. The experimental scheme of Example 1. Two plasmidspET_his_MLV_pD (encoding Moloney Murine Leukemia Virus (M-MLV) reversetranscriptase fused to protein D spacer) and pET_his_del_pD (encodinginactivated (57 amino acids deletion in pol domain) Moloney MurineLeukemia Virus (M-MLV) reverse transcriptase fused to protein D spacer)were used to synthesize PCR fragments. PCR fragments furtheron are usedin transcription reaction and synthesis of mRNA lacking STOP codon atthe 3′ end. Purified mRNA is mixed with ratio 1:50 =MLV (active RT):del(inactive RD and used for in vitro translation reaction. Duringtranslation reaction ribosomal complex synthesizes protein and stops atthe end of mRNA lacking STOP codon. Mixture of ternary complexes (TC) ispurified by ultracentrifugation on sucrose cushions. Purified ternarycomplexes (<3*10⁹ molecules taken) already containing mRNA linked to invitro translated MLV reverse transcriptase are used to prepare reversetranscription reaction mix supplemented with external dNTP set andprimer for RT reaction. Ice-cold RT reaction mixture is emulsifiedgiving ˜1*10¹⁰ water in oil compartments ˜2 μm in size. Emulsified RTreaction mixture (less than one TC (mRNA+MLV RT) per compartment isincubated for 1 hr at 42° C. in order to perform RT reaction. After thetemperature of compartmentalized RT reaction mixture is raised most ofTC dissociate releasing mRNA and reverse transcriptase. Successful RTreaction is performed only in compartments containing active MLV reversetranscriptase (MLV_pD) and no cDNA is synthesized in compartments withinactive reverse transcriptase (del_pD). Subsequent PCR amplifies cDNAand enrichment of active reverse transcriptase (MLV_pD) genes overinactive reverse transcriptase (del_pD) is observed.

FIG. 2. The structural scheme of pET_his_MLV_pD plasmid.

FIG. 3. Example 1—the agarose gel electrophoresis of first PCR performedon cDNA synthesized during CRD selection. Primers used: RD_Nde (SEQ IDNo: 9) and pD_55 (SEQ ID No: 10). Expected length of PCR fragments was2185 bp for MLV_pD and 2014 bp for del_pD. Amplification was analyzed on1% agarose gel loading 10 μl of PCR mix per well.

FIG. 4. Example 1—the agarose gel electrophoresis of nested PCR forpartial gene amplification performed on first PCR product. Primers used:M_F (SEQ ID No: 11) and M_2R (SEQ ID No: 12). Expected length of PCRfragments was 907 bp for MLV_pDa and 736 bp for del_pD. Amplificationwas analyzed on 1% agarose gel loading 10 μl of PCR mix per well.

FIG. 5. Example 1—the agarose gel electrophoresis of nested PCR for fullgene amplification performed on first PCR product. Primers used: M_Esp(SEQ ID No: 13) and M_Eri (SEQ ID No: 14). Expected length of PCRfragments was 2077 bp for MLV_pDa and 1906 bp for del_pD. Amplificationwas analyzed on 1% agarose gel loading 10 μl of PCR mix per well.

FIG. 6. The experimental scheme of CRD selection in Example 2. PCRfragments encoding mutants library of reverse transcriptase (in fusionwith protein D) MLV_pD was used to synthesize mRNA. Purified mRNA wasused for in vitro translation reaction. Ternary complexes (TC) ofmRNA-ribosome-MLV_pD (tRNA) were formed in translation mixture andstabilized by low temperature and high concentration of Mg²⁺ ions.Mixture of TC was purified by ultracentrifugation on sucrose cushions.Precipitated TC was dissolved in ice-cold buffer (50 mM Mg²⁺) and usedto prepare reverse transcription reaction mix supplemented with externaldNTP set and primer for RT reaction. Ice-cold RT reaction mixture wasemulsified giving ˜1*10¹⁰ water in oil compartments ˜2 μm in size.Optimal reaction temperature of MLV RT is ˜42° C. In order to select forreverse transcriptase variants, which are working better at highertemperatures emulsified RT reaction mixture (less than one TC (mRNA+MLVRT) per compartment) was incubated for 1 hr at 50° C. At thistemperature successful synthesis of full length cDNA was performedbetter in compartments containing more active or thermostable MLVreverse transcriptase variants. Subsequent PCR was used to amplify fulllength cDNA and enrichment of more active and thermostable reversetranscriptase genes was performed. By PCR amplified genes were movedback to CRD format restoring intact 5′ (START fragment—T7 polymerasepromoter, SD and his-tag coding sequences) and 3′ (END fragment—gslinker, protein D and second gs linker) sequences by ligation PCR.Reconstructed PCR fragment, containing enriched library of reversetranscriptase genes, was used for subsequent mRNA transcription and nextCRD selection round. Each selection round was performed at higher andhigher temperatures of RT reaction: 50° C. (1^(st) round); 52.5° C.(2^(nd) round); 55° C. (3^(rd) round); 57.5° C. (4^(th) round) and 60°C. (5^(th) round).

FIG. 7. The scheme of PCR fragment reconstruction before new round ofCRD selection. Mutated MLV RT library was digested with Esp3I (NcoIcompatible end) and EcoRI and ligated with START (244 bp) and END (398bp) fragments in order to get PCR fragment suitable for CRD selection.START fragment (containing T7 polymerase promoter, SD and his-tag codingsequences) was constructed by PCR amplification of initial 983 bp STARTfragment (target—plasmid pET_his_del_pD (SEQ ID No: 2),primers—pro-pIVEX (SEQ ID No: 3) and M_1R (SEQ ID No: 15)) andsubsequent digestion with NcoI (recognition sequence C↓CATGG) giving 244bp DNA fragment. END fragment (containing gs linker, protein D andsecond gs linker sequences) was constructed by PCR amplification ofinitial 1039 bp END fragment (target—plasmid pET_his_del_pD (SEQ ID No:2), primers—M_3F (SEQ ID No: 16) and pD-ter (SEQ ID No: 4)) andsubsequent digestion with EcoRI (recognition sequence G↓AATTC) giving398 bp DNA fragment.

FIG. 8. Reverse transcriptase activities of mutant RT variants measuredat 37° C., 50° C. and residual activity at 37° C. after 5 min incubationat 50° C. Reverse transcriptase activity at 37° C. is normalized to bealways 100% and is omitted. Thus only two types of columns (percents ofRT activity at 50° C. and residual RT activity at 37° C. after 5 minincubation at 50° C.) are shown. As a control is given wt M-MuLV reversetranscriptase used for mutants library construction. This primary enzymeis expressed in the same vector and purified in the same way as mutantvariants of RT. An average value of mutant RT activity at 50° C. for alltested mutants is about ˜92% and is more than 2 times higher comparingto wt enzyme (45%). An average residual activity of mutant RT variantsat 37° C. after 5 min preincubation at 50° C. is 12% (wt enzyme—11%).

FIG. 9. Specific activity (u/mg of protein) of partially purified wt andmutant RT variants measured 10 min at 37° C.

FIG. 10. The proposed experimental scheme of CRD selection using FACS.Protein of interest is displayed in ribosome display format. Purified,or just diluted many times, ternary complex comprisingmRNA-ribosome-protein (tRNA) is mixed with non fluorescent substrate (S)in reaction buffer and subsequently emulsified producing doublewater-in-oil-in-water emulsions. Active variants of compartmentalizedenzymes will convert substrate (S) to fluorescent product (P) allowingFACS to distinguish between fluorescent (active enzyme inside) and“dark” (inactive enzyme inside) droplets.

FIG. 11. The proposed experimental scheme for selection and evolution ofthermostable DNA polymerases using CRD. Polymerase of interest has to bedisplayed in ribosome display format. Optionally purified (or justdiluted many times) ternary complex comprising mRNA-ribosome-polymerasecan be used to prepare reaction mixture with reverse transcriptase(helper enzyme), dNTP's and primer set in PCR buffer. Reaction solutionshould be emulsified producing water-in-oil emulsion. In first RTstep—reverse transcriptase has to synthesize cDNA, which subsequentlywill serve as a target for second PCR step—cDNA amplification byribosome displayed DNA polymerase. One of the primers used in PCR canhave biotin for optional subsequent purification with streptavidin beadsand non-complementary 5′ end. After RT-PCR emulsions should be broken,newly synthesized DNA fragment can be purified via biotin andreamplified using new primer set, which will contain one primer withsequence non-complementary to cDNA, but identical to 5′ part of primerused in first amplification reaction (selective amplification of DNAover cDNA background). More active variants of DNA polymerase will beenriched over less active variants and can be used for further analysisor next selection round.

FIG. 12. The experimental scheme of Example 4. In this experimentalsetup M-MuLV reverse transcriptase is used as DNA dependent DNApolymerase. Two plasmids pET_his_MLV_D583N_pD (encoding RNase H minusMoloney Murine Leukemia Virus (M-MLV) reverse transcriptase fused toprotein D spacer) and pET_his_del_pD (encoding inactivated reversetranscriptase fused to protein D spacer—57 amino acids deletion in poldomain and point mutation D583N in RNase H domain) were used tosynthesize PCR fragments. PCR fragments furtheron are used intranscription reaction. Purified mRNA is mixed with ratio1:20=MLV_D583N_pD (active RT):del_pD (inactive RT) and used to preparemRNA/dsDNA complex by dsDNA ligation to mRNA mix using T4 DNA ligase.mRNA/dsDNA complex was used for in vitro translation reaction. Duringtranslation reaction ribosomal complex synthesizes protein and stops atthe end of mRNA (at the beginning of mRNA/DNA hybrid). Mixture ofternary complexes (TC) is purified by ultracentrifugation on sucrosecushions. Purified ternary complexes (<3*10⁹ molecules taken) alreadycontaining mRNA/dsDNA linked to in vitro translated polymerase (M-MuLVreverse transcriptase) are used to prepare elongation reaction mixsupplemented with external biotin-dUTP. Ice-cold reaction mixture isemulsified giving ˜1*10¹⁰ water in oil compartments μ2 μm in size.Emulsified elongation reaction mixture (less than one TC(mRNA/dsDNA+polymerase) per compartment is incubated for 30 min at 37°C. in order to incorporate biotinylated nucleotide. After thetemperature of compartmentalized reaction mixture is raised most of TCdissociate releasing mRNA/dsDNA complex and polymerase. Successfulincorporation reaction in to dsDNA substrate is performed only incompartments containing active polymerase (reversetranscriptase—MLV_D583N_pD) and no cDNA is synthesized in compartmentswith inactive polymerase (del_pD). After the emulsions are broken excessof biotin-dUTP is removed using gel-filtration mini-column. BiotinylatedmRNA/dsDNA complex is purified on streptavidin beads and used tosynthesize cDNA. Subsequent PCR amplifies cDNA and enrichment of activepolymerse (reverse transcriptase—MLV_D583N_pD) genes over inactivepolymerase (del_pD) is observed.

FIG. 13. Determination of biotin-dUTP incorporation efficiencies intomRNA/dsDNA complex and into self primed mRNA. Picture of RT-PCRperformed on samples of mRNA/dsDNA (MLV_D583N_pD) and mRNA (del_pD)after the incorporation of dTTP or biotin-dUTP. Predicted amplicons sizeare 907 bp for MLV_D583N_pD and 736 bp for del_pD cDNA. PCR productswere analyzed on 1% agarose gel loading 10 μl of PCR mix per well.

FIG. 14. General control of mRNA/dsDNA complex existence byincorporation of dTTP (biotin-dUTP) and [α-P³³]dATP. Radioactive dATPshould be introduced into dsDNA substrate subsequently after theincorporation of initial dTTP or biotin-dUTP. A ethidium bromidevisualized agarose gel (mRNA or mRNA/dsDNA bands ˜2.5 kb). B—the samegel as in A dried on filter paper and visualized using Cyclone PhosphorImager (Perkin-Elmer, Wellesley, Mass.). Labeled mRNA/dsDNA complexand/or only dsDNA bands are observed. C—structure and sequence of dsDNAcounterpart in mRNA/dsDNA complex SEQ ID NOS,: 21 and 22.

FIG. 15. The resultant final RT-PCR fragments analysis of Example 4.Predicted amplicons size are 907 bp for MLV_D583N_pD and 736 bp fordel_pD cDNA. PCR products were analyzed on 1% agarose gel loading 10 μlof PCR mix per well. RT-PCR samples before streptavidin beadscorresponds to the ratio of active and inactive polymerase genes 1:20(almost only del_pD fragment ˜736 bp is visibile). RT-PCR samples afterthe purification on streptavidin beads corresponds to the ratio ofactive and inactive polymerase genes after the single selectionround—1:1. An enrichment factor of ˜20 is observed in this selection.

FIG. 16. Some examples of alkaline agarose gels used to determinehighest temperature of 1 kb and 4.5 kb cDNA synthesis reaction. PCRmachine—Eppendor Mastercycle Gradient. A-D—1 kb cDNA synthesis (M-MuLV(wt), D200N, L603W and Q221R); temperature gradient 41.9° C., 43.6° C.,45.5° C., 47.8° C., 50.4° C., 53.1° C., 55.8° C., 58.1° C., 60.1° C.,62.1° C.; size standart—DNA Fast Ruler Middle Range (Fermentas). E-G—4.5kb cDNA synthesis (M2, M3 and M4); temperature gradient 49.8° C., 51.5°C., 53.4° C., 55.7° C., 58.3° C., 61.0° C., 63.7° C., 66.1° C., 68.0°C., 70.0° C.; size standart—Zip Ruler Express DNA ladder 2 (Fermentas).

FIG. 17. The CLUSTALW alignment of all 104 protein sequences without Nterminal His tag in order to have the same numeration of amino acids asis usually used in literature. Wild type sequence denoted as MLV (SEQ IDNo. 25) is always given as first sequence (mutated sequences representSEQ ID Nos: 26 to 128 based on the order in which they are shown).Mutations are marked using white font color in black background. Aminoacids positions, mutations of which somehow improve M-MuLV reversetranscriptase properties and are described in different patentapplications are marked in the alignment as columns of amino acids inbold font. Mutations originating from our selection and located in greycolumns indicate that our selection procedure precisely targeted thebeneficial hot spot or even exact amino acid mutations describedelsewhere. Sequences of analyzed proteins, activity of which at 50° C.was substantially better as compared to the primary wt M-MuLV (70% andmore as compared to 45% of wt activity) are highlighted withunderlining.

FIG. 18 Mutations frequency (in decreasing order) of selected RTvariants. Names of analyzed proteins, which activity at 50° C. wassubstantially better as compared to primary wt M-MuLV (70% and morecomparing to 45% of wt activity) are highlighted in black.

FIG. 19 Summarized table of data on M-MuLV (wt) reverse transcriptaseand single mutants, which contains: name of protein; selection frequency(number of sequenced mutants, which had exact mutation and the number inthe parentheses indicates total number of particular amino acidmutations found in selection); protein concentration (mg/ml); reversetranscriptase specific activity at 37° C. (u/mg); relative activity at50° C. (%); relative residual activity at 37° C. after 5 min enzymeincubation at 50° C. (%); specific RNase H activity of protein (u/mol);relative RNase H activity (%) and highest temperature of 1 kb cDNAsynthesis reaction.

FIG. 20 Summarized table of data on M MuLV (wt) reverse transcriptaseand single mutants, which contains: name of protein; proteinconcentration (mg/ml); reverse transcriptase specific activity at 37° C.(u/mg); relative activity at 50° C. (%) and highest temperature of 1 kband 4.5 kb cDNA synthesis reaction.

Appendix 1. The general scheme of mutations found in initial MLV RTlibrary (sequence between NcoI and EcoRI restriction sites—SEQ ID No.24). Twenty-three (23) nucleotide mutations were found among tensequenced genes (1 transversion, 20 transitions—mutated positions areunderlined, mutations indicated above the sequence, 2deletions—underlined and indicated as dashed line above the sequence)giving 15 amino acids exchanges, 6 silent mutations, 1 stop codon and 2frame shifts of coding frame—on average 1-2 amino acids substitutionsper gene.

Appendix 2. List of mutations found in all selected RT variants.Proteins are sorted by decreasing number of mutations.

Appendix 3. Sequence and information relating to SEQ ID Nos: 1 to 23.

EXAMPLE 1 CRD—Proof of Principle

To provide proof of principle for Compartmentalized Ribosome Displayselection system test selection was performed. Typical proof ofprinciple experiment should give positive signal for active enzyme (inour case RT-PCR fragment for original MLV reverse transcriptase) and nosignal for inactive enzyme (no RT-PCR fragment for inactivated MLVreverse transcriptase). A more sophisticated experiment is to usemixture of genes with defined ratio encoding active and inactiveenzymes. As a result of successful experiment genes encoding activeenzyme should be enriched over genes encoding inactive enzyme.

The general experimental scheme is shown in FIG. 1. Two plasmidspET_his_MLV_pD (encoding Moloney Murine Leukemia Virus (M-MLV) reversetranscriptase fused to protein D spacer) and pET_his_del_pD (encodinginactivated (57 amino acids deletion in pol domain) Moloney MurineLeukemia Virus (M-MLV) reverse transcriptase fused to protein D spacer)were used to synthesize PCR fragments. Further PCR fragments were usedin transcription reaction for synthesis of mRNA, which lacks STOP codonat the 3′ end. Purified mRNAs resulting from the two abovementioned PCRfragments were mixed with a ratio 1:50=MLV (active RT):del (inactive RT)and used for in vitro translation reaction. During translation reactionribosomal complex synthesizes protein and stops at the end of mRNAlacking STOP codon. Translation reaction was stopped by dilution withice-cold buffer containing 50 mM Mg²⁺. Low temperature, highconcentration of Mg²⁺ ions and absence of STOP codon at the end of mRNAstabilize ternary complexes (TC) of mRNA-ribosome-protein (tRNA).Mixture of ternary complexes (TC) was purified by ultracentrifugation onsucrose cushions. Ultracentrifugation was optimized in such a way thatTC (˜3.5 MDa) were precipitated at the bottom of ultracentrifugationtube, meanwhile small molecular weight molecules, proteins and most offree mRNA (˜0.9 MDa) remained in the supernatant. Precipitated TCs weredissolved in the ice-cold buffer (50 mM Mg²⁺). Purified ternarycomplexes (<3*10⁹ molecules taken) already containing mRNA linked to invitro translated MLV reverse transcriptase were used to prepare reversetranscription reaction mix supplemented with external dNTP set andprimer for RT reaction. Ice-cold RT reaction mixture was emulsifiedyielding ˜1*10¹⁰ water in oil compartments ˜2 μm in size. Emulsified RTreaction mixture (less than one TC (mRNA+MLV RT) per compartment wasincubated for 1 hr at 42° C. in order to perform RT reaction. After thetemperature of compartmentalized RT reaction mixture was raised most ofTCs dissociated releasing mRNA and reverse transcriptase. Successful RTreaction was performed only in compartments containing active MLVreverse transcriptase (MLV_pD) and no cDNA was synthesized incompartments with inactive reverse transcriptase (del_pD). SubsequentPCR ensured the amplification of synthesized cDNA and enrichment ofactive reverse transcriptase (MLV_pD) genes over inactive reversetranscriptase (del_pD) is observed.

Methods and Materials

Initial plasmid pET_his_MLV_pD (SEQ ID No: 1 and FIG. 2) was constructedby modification of pET type plasmid in T7 polymerase promoter andShine-Dalgarno sequences region and insertion of MLV H+ reversetranscriptase coding sequence (306-2363 on SEQ ID No: 1) with N-terminalHis-tag (258-305 on SEQ ID No: 1) and C-terminal fusion withglycine-serine (gs) linker (2364-2393 on SEQ ID No: 1), part of proteinD (pD) from phage lambda (2394-2669 on SEQ ID No: 1) and secondglycine-serine (gs) linker (2670-2759 on SEQ ID No: 1). N-terminalHis-tag is used for protein express purification. C-terminal fusion hasto remain in ribosome tunnel during protein in vitro translation andformation of mRNA-ribosome-MLV (tRNA) ternary complex.

M-MuLV reverse transcriptase has two main enzymatic activities: RNAdependent DNA polymerase and RNase H. Reverse transcriptase RNase Hactivity was turned off introducing point mutation D583N (singlenucleotide G to A exchange at position 2055 in plasmid pET_his_MLV_pD,SEQ ID No: 1). Aspartate 583 is located in RNase H active site, isinvolved in Mg ion binding and is crucial for RNase H activity. Newplasmid is identified as pET_his_MLV_D583N_pD and was used for furtherconstruction of next plasmid pET_his_del_pD (SEQ ID No: 2), whichencodes inactivated reverse transcriptase. Plasmid pET_his_MLV_D583N_pDwas digested with restriction endonuclease XmaJI (recognition sequenceC↓CTAGG—positions 1047 and 1218 on SEQ ID No: 1). Gene fragment 171 bpin length was removed and digested plasmid was self-ligated, yieldingplasmid pET_his_del_pD (SEQ ID No: 2), which encodes reversetranscriptase gene shorter by 171 nucleotides or 57 amino acids, withoutshift in protein translation frame.

It was important to have the same reverse transcriptase gene: 1) shorterin length (for easy PCR detection); 2) inactive (it was confirmedexperimentally that deletion of 57 amino acids in polymerase domaincompletely inactivated polymerase activity and mutation D583Ninactivated RNase H activity); and 3) without frameshift (any frameshiftwill result in appearance of STOP codons, which are not compatible withribosome display format).

Preparation of PCR fragments for in vitro transcription. PCR mixture wasprepared on ice: 20 μl-10× Taq buffer with KCl (Fermentas); 20 μl-2 mMof each dNTP (Fermentas); 12 μl-25 mM MgCl₂ (Fermentas); 16 μl—DMSO(D8418-Sigma); 4 μl-1 u/μl LC (recombinant) Taq DNA Polymerase(Fermentas); 1 μl-100 μM pro-pIVEX primer (SEQ ID No: 3); 1 μl-100 μMpD-ter primer (SEQ ID No: 4); 122 μl water—mixture divided into twotubes 2×98 μl. To 2×98 μl of PCR master mix were added either 2 μl ofpET_his_MLV_pD (diluted to ˜1 ng/μl) or 2 μl of pET_his_del_pD (dilutedto ˜1 ng/μl). The cycling protocol was: initial denaturation step 3 minat 94° C., 30 cycles (45 sec at 94° C., 45 sec at 53° C., and 2 min at72° C.) and final elongation 5 min at 72° C. Amplification was ˜7000fold from 2 ng of plasmid (7873 bp) target to ˜5 μg (50 ng/μl) ofamplified product (2702 bp PCR fragment for pET_his_MLV_pD; 2531 bp PCRfragment for pET_his_del_pD).

Transcription mixture was prepared: 40 μl-5×T7 transcription buffer (1 MHEPES-KOH pH 7.6; 150 mM Mg acetate; 10 mM spermidin; 0.2 M DTT); 56μl-25 mM of each NTP (Fermentas); 8 μl-20 u/μl T7 RNA polymerase(Fermentas); 4 μl-40 u/μl RiboLock RNase inhibitor (Fermentas); 52 μlnuclease-free water—mixture divided into two tubes 2×80 μl and add 20μl-50 ng/μl of MLV_pD(pro-pIVEX//pD-ter) or 20 μl-50 ng/μl ofdel_pD(pro-pIVEX//pD-ter) PCR mixture. Transcription was performed 3 hrat 37° C.

Both transcription mixtures were diluted to 200 μl with ice-coldnuclease-free water and 200 μl of 6 M LiCl solution was added. Mixtureswere incubated 30 min at +4° C. and centrifuged for 30 min at +4° C. incooling centrifuge at max speed (25′000 g). Supernatant was discardedand RNA pellet washed with 500 μl of ice-cold 75% ethanol. Tubes againwere centrifuged for five min at +4° C. in cooling centrifuge at maxspeed and supernatant was discarded. RNA pellet was dried for 12 min atroom temperature and subsequently resuspended in 200 μl of nuclease-freeice-cold water by shaking for 15 min at +4° C. and 1400 rpm. Tubes againwere centrifuged for five min at +4° C. in cooling centrifuge at maxspeed in order to separate not dissolved RNA. About 180 μl ofsupernatant were moved to new tube with 20 μl of 10× DNase I buffer(Mg²⁺) (Fermentas); 1 μl-1 u/μl DNaseI (RNase-free) (Fermentas) andincubated for 20 min at +37° C. in order to degrade DNA. To each tubewere added 20 μl of 3 M sodium acetate pH 5.0 solution and 500 μl ofice-cold 96% ethanol. Finally RNA was precipitated by incubation for 30min at −20° C. and centrifugation for 30 min at +4° C. in coolingcentrifuge at max speed (25,000 g). Supernatant was discarded and RNApellet washed with 500 μl of ice-cold 75% ethanol. Tubes again werecentrifuged for 5 min at +4° C. in cooling centrifuge at max speed andsupernatant was discarded. RNA pellet was dried for 12 min at roomtemperature and subsequently resuspended in 43 μl of nuclease-freeice-cold water by shaking for 15 min at +4° C. and 1400 rpm. RNAsolution was aliquoted 4×10 μl and liquid nitrogen frozen. Concentrationof mRNA was measured spectrophotometrically and double checked onagarose gel using RiboRuler™ RNA Ladder, High Range (Fermentas)—MLV_pDmRNA ˜1.2 μg/μl; del_pD mRNA ˜1.2 μg/μl.

Purified mRNA is mixed with ratio 1:50=MLV (active RT):del (inactiveRT). MLV_pD mRNA was diluted 25 times to ˜48 ng/μl and 1 μl (˜48 ng) wasmixed with 2 μl˜1.2 μg/μl del_pD mRNA (2.4 μg) giving mRNA mixture ˜0.8μg/μl with ratio 1:50. In vitro translation was performed using twotranslation systems RTS 100 E. coli HY Kit (03 186 148 001—Roche) andsynthetic WakoPURE (295-59503—Wako). Proteins translation sequences aregiven in SEQ ID No: 6 for MLV_pD and SEQ ID No: 7 for del_pD.

Translation mixture for RTS HY system (25 μl): 6 μl- E. coli lysate(Roche); 5 μl—Reaction Mix (Roche); 6 μl—amino acids (Roche); 0.5 μl-100mM Met (Roche); 0.5 μl-40 u/l RiboLock RNase inhibitor (Fermentas); 0.4μl-200 μM assrA oligonucleotide (SEQ ID No: 5); 0.25 μl-1 M DTT; 2.5 μlreconstitution buffer (Roche); 2.5 μl nuclease-free water and 1.5 μl-0.8μg/μl mRNA mixture 1:50=MLV_pD:del_pD (˜1200 ng). In vitro translationwas performed for 20 min at 30° C.

Translation mixture for WakoPURE system (˜25 μl): 12.5 μl—A solution(Wako); 5 μl—B solution (Wako); 0.5 μl-40 u/μl RiboLock RNase inhibitor(Fermentas); 0.4 μl-200 μM assrA oligonucleotide (SEQ ID No: 6); 0.25μl-1 M DTT; 5 μl nuclease-free water and 1.5 μl-0.8 μg/μl mRNA mixture1:50=MLV_pD:del_pD (˜1200 ng). In vitro translation was performed for 30min at 37° C.

Both translations (˜25 μl) were stopped by adding 155 μl of ice-coldstopping buffer WBK₅₀₀+DTT+triton (50 mM tris-acetate pH 7.5 at 25° C.;50 mM NaCl; 50 mM Mg-acetate; 500 mM KCl; 10 mM DTT; 0.1% (v/v)—tritonx-100 (T8787-Sigma)) and centrifuged for 5 min at +4° C. and 25,000 g.Very carefully 160 μl of centrifuged translation mixture was pippeted onthe top of 840 μl 35% (w/v) sucrose solution in WBK₅₀₀+DTT+triton (50 mMtris-acetate pH 7.5 at 25° C.; 50 mM NaCl; 50 mM Mg-acetate; 500 mM KCl;10 mM DTT; 0.1% (v/v)—triton x-100 (T8787-Sigma); 35% (w/v)—sucrose(84097-Fluka)). In order to purify ternary complexes (TC) ofmRNA-ribosome-protein (tRNA) ultracentrifugation was performed usingTL-100 Beckman ultracentrifuge; TLA100.2 fixed angle rotor (Beckman);transparent 1 ml ultracentrifugation tubes (343778-Beckman) for 9 min at+4° C. and 100,000 rpm. In order to keep small transparent pellet of TCat the bottom of ultracentrifugation tube intact tubes were handled withcare. Initially 750 μl of solution was removed from the very top of thecentrifugation tube. Then very carefully tube walls were washed with 750μl of WBK₅₀₀ (50 mM tris-acetate pH 7.5 at 25° C.; 50 mM NaCl; 50 mMMg-acetate; 500 mM KCl). Finally all solution was removed starting fromthe very top of the centrifugation tube and the pellet was dissolved in30 μl of ice-cold stopping buffer WBK₅₀₀+DTT+triton (50 mM tris-acetatepH 7.5 at 25° C.; 50 mM NaCl; 50 mM Mg-acetate; 500 mM KCl; 10 mM DTT;0.1% (v/v)—triton x-100 (T8787-Sigma)).

As it was determined using radioactively labeled mRNA afterultracentrifugation 5%-30% of input mRNA is located in ternary complexpellet. Therefore it was expected to have less than 360 ng (30% from1200 ng mRNA used in translation reaction) of mRNA in 30 μl of buffer(˜12 ng/μl or 9*10⁹ molecules/μl of ternary complex).

Reverse transcription reaction mixture for selection was prepared onice: 60 μl-5× reaction buffer for Reverse Transcriptase (Fermentas); 7.5μl-40 u/μl RiboLock RNase inhibitor (Fermentas); 15 μl-20 μM pD_42oligonucleotide (SEQ ID No: 8); 188 μl nuclease-free water—mixturedivided into two tubes 2×135 μl and 0.9 μl of purified (<8*10⁹molecules) TC (translation in Roche—RTS HY kit) or 0.9 μl of purified(<8*10⁹ molecules) TC (translation in Wako—WakoPURE) were added. Eachreaction mixture (˜135 μl) again was divided into two tubes 45 μl and 90μl. To the first part—45 μl of RT mixture 5 μl of nuclease-free waterwere added. This sample is considered to be the negative selectioncontrol (without dNTP) and has to prove that there is no DNAcontamination in the reaction mixture and cDNA synthesis is strictlylinked to reverse transcriptase functional activity coming from MLV RTin ternary complex. To the second part—90 μl of RT mixture 10 μl—10 mMeach dNTP Mix (Fermentas) were added and reaction mixture again wasdivided into two tubes—50 μl for selection control and 50 μlsupplemented with 1 μl-200 u/μl of RevertAid H- M-MuLV ReverseTranscriptase (Fermentas) for positive selection control. According tothe protocol each reverse transcription reaction mix contains <2.7*10⁹molecules of ternary complex in 50 μl volume.

Oil-surfactant mixture for emulsification was prepared by mixing ABIL EM90 (Goldschmidt) into mineral oil (M5904-Sigma) to final concentrationof 4% (v/v) (Ghadessy and Holliger, 2004; U.S. 2005/064460). Emulsionswere prepared at +4° C. in 5 ml cryogenic vials (430492-Corning) bymixing 950 μl of oil-surfactant mixture with 50 μl of RT mixture. Mixingwas performed using MS-3000 magnetic stirrer with speed control (Biosan)at ˜2100 rpm; Rotilabo®—(3×8 mm) magnetic followers with centre ring(1489.2-Roth); water phase was added by 10 μl aliquots every 30 sec,continuing mixing for two more minutes (total mixing time—4 min).According to optical microscopy data compartments in our emulsions varyfrom 0.5 μm to 10 μm in size with average diameter of ˜2 μm. Thereforeit was expected to have ˜1*10¹⁰ water in oil compartments after theemulsification of 50 μl reverse transcription reaction mixture, whichcontains less than 2.7*10⁹ molecules of ternary complex (about 1 mRNAand reverse transcriptase molecules per 3-4 compartments).

All six emulsions representing RT reactions with TC, translation inRoche—RTS HY kit (negative selection control, selection control andpositive selection control) and with TC, translation in Wako—WakoPURE(negative selection control, selection control and positive selectioncontrol) were incubated 1 hr at +42° C.

To recover the reaction mixtures emulsions were moved to 1.5 ml tube,centrifuged for one min at room temperature and 25,000 g. Oil phase wasremoved leaving concentrated but still intact emulsion at the bottom ofthe tube and 250 μl of PB buffer (Qiagen PCR purification kit) wereadded. Finally emulsions were broken by extraction with 0.9 mlwater-saturated ether; 0.9 ml water-saturated ethyl-acetate (in order toremove ABIL EM 90 detergent) and again 0.9 ml water-saturated ether.Water phase was dried for 5 min under vacuum at room temperature.Synthesized cDNA was purified with Qiagen PCR purification kit andeluted in 30 μl of EB buffer (Qiagen PCR purification kit).

Amplification of cDNA was performed by nested PCR. Initial PCR mixturewas prepared on ice: 16 μl-10× Taq buffer with KCl (Fermentas); 16 μl-2mM of each dNTP (Fermentas); 9.6 μl-25 mM MgCl₂ (Fermentas); 3.2 μl- 1u/μl LC (recombinant) Taq DNA Polymerase (Fermentas); 0.8 μl-100 μMRD_Nde primer (SEQ ID No: 9); 0.8 μl-100 μM pD_55 primer (SEQ ID No:10); 74 μl water—mixture was divided into 6 samples×15 μl (6×15 μl) and30 μl. To 6×15 μl of PCR master mix were added 5 μl of cDNA (1-6 RTsamples); to 30 μl of PCR master mix were added 9 μl water and mixtureagain divided into two tubes 2×19.5 μl for negative PCR control (plus0.5 μl water) and positive PCR control (plus 0.5 μl—1:1 mixture (˜1 ng)of pET_his_MLV_pD and pET_his_del_pD plasmids). The cycling protocolwas: initial denaturation step 3 min at 94° C., 25 cycles (45 sec at 94°C., 45 sec at 58° C., and 2 min at 72° C.) and final elongation 5 min at72° C. Expected length of PCR fragments was 2185 bp for MLV_pD and 2014bp for del_pD. Amplification was analyzed on 1% agarose gel loading 10μl of PCR mix per well (FIG. 3).

Nested PCR was performed using two different sets of primers, givingeither partial gene amplification (for better resolution of MLV:del cDNAratio in RT samples) or full gene amplification (to prove possibility offull gene recovery).

Nested PCR mixture for partial gene amplification was prepared on ice:28 μl-10× Taq buffer with KCl (Fermentas); 28 μl-2 mM of each dNTP(Fermentas); 16.8 μl-25 mM MgCl₂ (Fermentas); 5.6 μl-1 u/μl LC(recombinant) Taq DNA Polymerase (Fermentas); 1.4 μl-100 μM M_F primer(SEQ ID No: 11); 1.4 μl-100 μM M_2R primer (SEQ ID No: 12); 185 μlwater—mixture divided 2×19 μl and 6×38 μl. To 2×19 μl of PCR master mixwas added 1 μl of positive or negative controls of first PCR (primersset RD_Nde//pD_55)—30 PCR cycles amplification; to 6×38 μl of PCR mastermix were added 2 μl of first PCR (primers set RD_Nde//pD_55) (1-6samples)—each sample again was divided into two 2×20 μl for 23 or 30 PCRcycles amplification. The cycling protocol was: initial denaturationstep 3 min at 94° C., 23 or 30 cycles (45 sec at 94° C., 45 sec at 57°C., and 1 min at 72° C.) and final elongation 5 min at 72° C. Expectedlength of PCR fragments was 907 bp for MLV_pD and 736 bp for del_pD.Amplification was analyzed on 1% agarose gel loading 10 μl of PCR mixper well (FIG. 4).

Nested PCR mixture for full gene amplification was prepared on ice: 28μl-10× Taq buffer with KCl (Fermentas); 28 μl-2 mM of each dNTP(Fermentas); 16.8 μl-25 mM MgCl₂ (Fermentas); 5.6 μl-1 u/μl LC(recombinant) Taq DNA Polymerase (Fermentas); 1.4 μl-100 μM M_Esp primer(SEQ ID No: 13); 1.4 μl-100 μM M_Eri primer (SEQ ID No: 14); 185 μlwater—mixture divided 2×19 μl and 6×38 μl. To 2×19 μl of PCR master mixwas added 1 μl of positive or negative controls of first PCR (primersset RD_Nde//pD_55)—30 PCR cycles amplification; to 6×38 μl of PCR mastermix were added 2 μl of first PCR (primers set RD_Nde//pD_55) (1-6samples)—each sample again was divided into 2×20 μl for 23 or 30 PCRcycles amplification. The cycling protocol was: initial denaturationstep 3 min at 94° C., 23 or 30 cycles (45 sec at 94° C., 45 sec at 55°C., and 2 min at 72° C.) and final elongation 5 min at 72° C. Expectedlength of PCR fragments was 2077 bp for MLV_pD and 1906 bp for del_pD.Amplification was analyzed on 1% agarose gel loading 10 μl of PCR mixper well (FIG. 5).

Results

To demonstrate proof of principle for Compartmentalized Ribosome Display(CRD) method selection was performed using starting 1:50=MLV:del mixtureof two mRNA encoding active (MLV) and inactive (del) reversetranscriptases fused to protein D spacer (FIG. 1). In vitro translationwas performed using two different translation systems Roche—RTS 100 E.coli HY or Wako—WakoPURE in order to understand which translation systemis better in our experimental setup. For each translation system threecompartmentalized RT reactions were performed: negative selectioncontrol without dNTP, which has to prove that there is no DNAcontamination in the reaction mixture; selection control, which has todemonstrate the enrichment of genes encoding active (MLV) reversetranscriptase over genes encoding inactivated enzyme (del), because onlyactive enzyme can synthesize cDNA; and positive selection controlsupplemented with external RevertAid H- commercial reversetranscriptase, which has to serve as positive RT control synthesizingcDNA from both MLV_pD and del_pD mRNA in all compartments, showing realratio of genes in reaction mixture without selection pressure applied.

Synthesyzed cDNA was amplified by nested PCR. The picture of agarose gelelectrophoresis of initial PCR (25 cycles) (FIG. 3) showed weak PCRfragments bands only in case of both positive selection controls(translation systems—Roche and Wako). This was normal, because thesesamples contained external RT enzyme, which synthesizes cDNA much moreefficiently comparing to reactions containing only one in vitrosynthesized reverse transcriptase molecule per compartment.

The picture of agarose gel electrophoresis of nested PCR (partial geneamplification) is shown in FIG. 4. Amplification results after 23 and 30PCR cycles are consistent:

1) there is no amplification (no DNA contamination) in negativeselection controls (w/o dNTP);

2) very efficient amplification of del_pD cDNA (736 bp DNA fragment) isobserved in positive selection controls (external RT enzyme) and noMLV_pD cDNA amplification is visible, because initial ratio of MLV_pD todel_pD mRNA is 1:50;

3) amplification of both cDNA MLV_pD (907 bp DNA fragment) and del_pD(736 bp DNA fragment) are observed in case of selection controls;

4) ratio of MLV_pD:del_pD˜1:1 is observed in case of reversetranscriptase synthesized by Roche in vitro translation system, whatmeans ˜50 times enrichment of MLV_pD genes over del_pD genes startingfrom initial 1:50 ratio;

5) ratio of MLV_pD:del_pD˜1:3 is observed in case of reversetranscriptase synthesized by Wako in vitro translation system, whatmeans ˜16 times enrichment of MLV_pD genes over del_pD genes startingfrom initial 1:50 ratio.

The picture of agarose gel electrophoresis of nested PCR (full geneamplification) is shown in FIG. 5. Amplification results after 23 and 30PCR cycles was consistent in between and comparing to results of nestedPCR used for partial gene amplification (FIG. 5):

1) there is no amplification (no DNA contamination) in negativeselection controls (w/o dNTP);

2) very efficient amplification of del_pD cDNA (1906 bp DNA fragment) isobserved in positive selection controls (external RT enzyme) and noMLV_pD cDNA amplification is visible, because initial ratio of MLV_pD todel_pD mRNA is 1:50;

3) amplification of both cDNA MLV_pD (2077 bp DNA fragment) and del_pD1906 bp DNA fragment) are observed in case of selection controls;

4) it is difficult to determine ratio of MLV_pD:del_pD in case of fullgene amplification, because relative difference between 2077 bp (MLV_pD)and 1906 bp (del_pD) DNA fragments is not big enough, but in generalratios are similar to results of nested PCR used for partial geneamplification.

As a result of this example we can conclude that during reversetranscription reaction performed in CRD format we have enriched genesencoding active MLV reverse transcriptase over the genes encodinginactive enzyme by a factor of 50 in the case of Roche translationsystem, or by a factor of 16 in case of Wako translation system used tosynthesize enzymes in vitro.

EXAMPLE 2 CRD—Selection for Reverse Transcriptase, Which Shows ImprovedPerformance at Higher Temperatures

To understand how efficiently the Compartmentalized Ribosome Display(CRD) selection worked, an evolution experiment of Moloney MurineLeukemia Virus reverse transcriptase (M-MuLV RT) was performed. Thegeneral experimental scheme is shown in FIG. 6. Initial mutants libraryof reverse transcriptase was constructed by error-prone PCR usingnucleotide analogues dPTP and 8-oxo-dGTP. Whole gene (˜2 kb) mutagenesiswas performed introducing 2-3 nucleotides or 1-2 amino acids mutationsper gene. PCR fragment encoding mutants library of reverse transcriptase(in fusion with protein D) MLV_pD was used to synthesize mRNA. PurifiedmRNA was used for in vitro translation reaction. Ternary complexes (TC)of mRNA-ribosome-MLV_pD (tRNA) were formed in translation mixture andstabilized by low temperature and high concentration of Mg²⁺ ions.Mixture of TC was purified by ultracentrifugation on sucrose cushions.Precipitated TC was dissolved in ice-cold buffer (50 mM Mg²⁺) and usedto prepare reverse transcription reaction mix supplemented with externaldNTP set and primer for RT reaction. Ice-cold RT reaction mixture wasemulsified giving ˜1*10¹⁰ water in oil compartments ˜2 μm in size.Optimal reaction temperature of MLV RT is ˜42° C. In order to select forreverse transcriptase variants, which are working better at highertemperatures, emulsified RT reaction mixture (less than one TC (mRNA+MLVRT) per compartment) was incubated for 1 hr at 50° C. At thistemperature successful synthesis of full length cDNA was performedbetter in compartments containing more active or thermostable MLVreverse transcriptase variants. Subsequent PCR was used to amplify fulllength cDNA and enrichment for more active and thermostable reversetranscriptase genes was performed. By PCR amplified genes were movedback to CRD format restoring intact 5′ (START fragment—T7 polymerasepromoter, SD and his-tag coding sequences) and 3′ (END fragment—gslinker, protein D and second gs linker) sequences by ligation-PCR.

Reconstructed PCR fragment, containing enriched library of reversetranscriptase genes, was used for subsequent mRNA transcription and nextCRD selection round. Each selection round was performed at higher andhigher temperatures of RT reaction: 50° C. (1^(st) round); 52.5° C.(2^(nd) round); 55° C. (3^(rd) round); 57.5° C. (4^(th) round) and 60°C. (5^(th) round).

Amplified library of reverse transcriptase genes (without C terminal pDlinker) after 5^(th) selection round was cloned into plasmid vector.Individual clones were sequenced and analyzed. Pool of evolved proteinsas well as individual mutants were purified via his-tag using affinitychromatography. MLV reverse transcriptase specific activities at 37° C.,50° C. and residual activity at 37° C. after 5 min enzyme incubation at50° C. were determined.

Methods and Materials

Initial plasmid pET_his_MLV_pD (SEQ ID No: 1 and FIG. 2) was used as astarting material for error-prone PCR. Mutations were introduced usingnucleotide analogues dPTP and 8-oxo-dGTP. PCR mixture for error pronePCR was prepared on ice: 10 μl-10× Taq buffer with KCl (Fermentas); 10μl-2 mM of each dNTP (Fermentas); 6 μl-25 mM MgCl₂ (Fermentas); 2 μl-1u/μl LC (recombinant) Taq DNA Polymerase (Fermentas); 0.5 μl-100 μMM_Esp primer (SEQ ID No: 13); 0.5 μl-100 μM M_Eri primer (SEQ ID No:14); 1 μl-10 μM dPTP (TriLink BioTechnolgies); 5 μl-100 μM 8-oxo-dGTP(TriLink BioTechnolgies); 3.75 μl-40 ng/μl (totally 150 ng) ofpET_his_MLV_pD plasmid; 61.25 μl water. The cycling protocol was:initial denaturation step 3 min at 94° C., 30 cycles (30 sec at 94° C.,30 sec at 55° C., and 2 min at 72° C.) and final elongation 5 min at 72°C. Amplification was 150-300 fold from 150 ng of plasmid (7873 bp)target to ˜6-12 μg of amplified product (2077 bp PCR fragment forpET_his_MLV_pD). PCR fragment was purified using Qiagen PCR purificationkit, digested with Esp3I (recognition sequence CGTCTC (1/5)) and EcoRI(recognition sequence G↓AATTC) and finally purified from agarose gelusing Qiagen Gel extraction kit giving DNA concentration ˜50 ng/μl.

Mutagenesis efficiency and library quality was checked by sequencing ofindividual clones subcloned back into original pET_his_MLV_pD plasmiddigested with NcoI and EcoRI. As expected mutations were distributedrandomly all over the amplified sequence of MLV RT gene (Appendix 1).Among 10 sequenced genes 23 nucleotide mutations (1 transversion, 20transitions, 2 deletions—labeled red in Appendix 1) were found giving 15amino acids exchanges, 6 silent mutations, 1 stop codon and 2 frameshifts of coding frame—on average 1-2 amino acids substitutions pergene.

Mutated library was ligated with START (244 bp) and END (398 bp)fragments in order to get PCR fragment suitable for CRD selection (FIG.7). START fragment (containing T7 polymerase promoter, SD and his-tagcoding sequences) was constructed by PCR amplification of initial 983 bpSTART fragment (target—plasmid pET_his_del_pD (SEQ ID No: 2),primers—pro-pIVEX (SEQ ID No: 3) and M_1R (SEQ ID No: 15)) andsubsequent digestion with NcoI (recognition sequence C↓CATGG) giving 244bp DNA fragment. END fragment (containing gs linker, protein D andsecond gs linker sequences) was constructed by PCR amplification ofinitial 1039 bp END fragment (target—plasmid pET_his_del_pD (SEQ ID No:2), primers—M_3F (SEQ ID No: 16) and pD-ter (SEQ ID No: 4)) andsubsequent digestion with EcoRI (recognition sequence G↓AATTC) giving398 bp DNA fragment.

Ligation reaction (150μl) was prepared at room temperature: 15 μl-10×ligation buffer for T4 DNA Ligase (Fermentas); 15 μl-1 u/μl T4 DNAligase (Fermentas); 26 μl-50 ng/μl mutated MLV RT library digested withEsp3I (NcoI compatible end) and EcoRI (˜1300 ng or ˜5.9*10¹¹ molecules);9.4 μl-35 ng/μl START fragment digested with NcoI (˜329 ng or ˜1.2*10¹²molecules); 15.7 μl-35 ng/μl END fragment digested with EcoRI (˜548 ngor ˜1.2*10¹² molecules); 68.9 μl—water. Ligation was performed overnightat +4° C. Reaction mixture was treated once with phenol and twice withchloroform, precipitated and dissolved in 53 μl of water. Approximateligation yield ˜20% was determined comparing amplification efficiency ofligation mixture and known amount of plasmid pET_his_MLV_pD. Taking intoaccount 20% ligation yield diversity of MLV RT mutants library wasdefined as ˜1.2*10¹¹ molecules (50 μl).

Ligated MLV RT library was amplified by PCR (1 ml—prepared on ice): 100μl-10× Taq buffer with KCl (Fermentas); 100 μl-2 mM of each dNTP(Fermentas); 60 μl-25 mM MgCl₂ (Fermentas); 80 μl—DMSO (D8418-Sigma); 20μl-1 u/μl LC (recombinant) Taq DNA Polymerase (Fermentas); 5 μl-100 μMpro-pIVEX primer (SEQ ID No: 3); 5 μl-100 μM pD-ter-primer (SEQ ID No:17); 20 μl—ligated MLV RT library (˜5*10¹⁰ molecules); 610 μl—water. Thecycling protocol was: initial denaturation step 3 min at 94° C., 15cycles (30 sec at 94° C., 30 sec at 53° C., and 3 min at 72° C.) andfinal elongation 5 min at 72° C. Amplification was ˜200 fold from˜5*10¹⁰ molecules (corresponds to ˜150 ng) of final ligation fragment2702 bp in size target to ˜30 μg (30 ng/μl) of amplified product (2702bp PCR fragment).

1^(st) Selection Round

Transcription mixture (100 μl) was prepared: 20 μl-5× T7 transcriptionbuffer (1 M HEPES-KOH pH 7.6; 150 mM Mg acetate; 10 mM spermidin; 0.2 MDTT); 28 μl-25 mM of each NTP (Fermentas); 4 μl-20 u/μl T7 RNApolymerase (Fermentas); 2 μl-40 u/μl RiboLock RNase inhibitor(Fermentas); 30 μl-30 ng/μl of mutants library (pro-pIVEX//pD-ter-) PCRmixture (˜900 ng or ˜3*10¹¹ molecules); 16 μl nuclease-free water.Transcription was performed 3 hr at 37° C. (library diversity ˜5*10¹⁰molecules).

Transcription mixture was diluted to 200 μl with ice-cold nuclease-freewater and 200 μl of 6 M LiCl solution were added. Mixture incubated 30min at +4° C. and centrifuged for 30 min at +4° C. in cooling centrifugeat max speed (25′000 g). Supernatant was discarded and RNA pellet washedwith 500 μl of ice-cold 75% ethanol. The tube again was centrifuged for5 min at +4° C. in cooling centrifuge at max speed and supernatant wasdiscarded. RNA pellet was dried for 12 min at room temperature andsubsequently resuspended in 200 μl of nuclease-free ice-cold water byshaking for 15 min at +4° C. and 1400 rpm. The tube again wascentrifuged for 5 min at +4° C. in cooling centrifuge at max speed inorder to separate not dissolved RNA. About 180 μl of supernatant weremoved to new tube with 20 μl of 10× DNase I buffer (Mg²⁺) (Fermentas); 1μl-1 u/μl DNaseI (RNase-free) (Fermentas) and incubated for 30 min at+37° C. in order to degrade DNA. To the reaction mixture were added 20μl of 3 M sodium acetate pH 5.0 solution and 500 μl of ice-cold 96%ethanol. Finally RNA was precipitated by incubation for 30 min at −20°C. and centrifugation for 30 min at +4° C. in cooling centrifuge at maxspeed (25,000 g). Supernatant was discarded and RNA pellet washed with500 μl of ice-cold 75% ethanol. The tube again was centrifuged for 5 minat +4° C. in cooling centrifuge at max speed and supernatant wasdiscarded. RNA pellet was dried for 12 min at room temperature andsubsequently resuspended in 33 μl of nuclease-free ice-cold water byshaking for 10 min at +4° C. and 1400 rpm. The RNA solution wasaliquoted 3×10 μl and liquid nitrogen frozen. The mRNA concentration wasmeasured spectrophotometrically and double checked on agarose gel usingRiboRuler™ RNA Ladder, High Range (Fermentas)—MLV RT library mRNA ˜2.1μg/μl.

In vitro translation was performed using RTS 100 E. coli HY (03 186 148001- Roche) translation system (25 μl): 6 μl-E. coli lysate (Roche); 5μl—Reaction Mix (Roche); 6 μl—amino acids (Roche); 0.5 μl-100 mM Met(Roche); 0.5 μl-40 u/μl RiboLock RNase inhibitor (Fermentas); 0.4 μl-200μM assrA oligonucleotide (SEQ ID No: 5); 0.25 μl-1 M DTT; 3 μlreconstitution buffer (Roche); 2.5 μl nuclease-free water and 0.6 μl-2.1μg/μl mRNA (1200 ng). The reaction mixture was incubated for 20 min at30° C. Translation was stopped adding 155 μl of ice-cold stopping bufferWBK₅₀₀+DTT+triton (50 mM tris-acetate pH 7.5 at 25° C.; 50 mM NaCl; 50mM Mg-acetate; 500 mM KCl; 10 mM DTT; 0.1% (v/v)—triton x-100(T8787-Sigma)) and centrifuged for 5 min at +4° C. and 25′000 g. Verycarefully 160 μl centrifuged translation mixture was pippeted on the topof 840 μl 35% (w/v) sucrose solution in WBK₅₀₀+DTT+Triton (50 mMtris-acetate pH 7.5 at 25° C.; 50 mM NaCl; 50 mM Mg-acetate; 500 mM KCl;10 mM DTT; 0.1% (v/v)—triton x-100 (T8787-Sigma); 35% (w/v)—sucrose(84097-Fluka)). In order to purify ternary complexes (TC) ofmRNA-ribosome-MLV (tRNA) ultracentrifugation was performed using TL-100Beckman ultracentrifuge; TLA100.2 fixed angle rotor (Beckman);transparent 1 ml ultracentrifugation tubes (343778-Beckman) for 9 min at+4° C. and 100′000 rpm. In order to keep small transparent pellet of TCat the bottom of ultracentrifugation tube intact tubes were handled withcare. Initially 750 μl of solution was removed from the very top of thecentrifugation tube. Then very carefully tube walls were washed with 750μl of WBK₅₀₀ (50 mM tris-acetate pH 7.5 at 25° C.; 50 mM NaCl; 50 mMMg-acetate; 500 mM KCl). Finally all solution was removed starting fromthe very top of the centrifugation tube and pellet was dissolved in 30μl of ice-cold stopping buffer WBK₅₀₀+DTT+triton (50 mM tris-acetate pH7.5 at 25° C.; 50 mM NaCl; 50 mM Mg-acetate; 500 mM KCl; 10 mM DTT; 0.1%(v/v)—triton x-100 (T8787-Sigma)).

As it was determined experimentally, using radioactively labeled mRNA,ultracentrifugation yielded 5%-30% of input mRNA in ternary complexpellet. Therefore it was expected to have less than 360 ng (30% from1200 ng mRNA used in translation reaction) of mRNA in 30 μl of buffer(˜12 ng/μl or 9*10⁹ molecules/μl of ternary complex).

Reverse transcription reaction mixture for selection was prepared onice: 60 μl-5× reaction buffer for Reverse Transcriptase (Fermentas); 7.5μl-40 u/μl RiboLock RNase inhibitor (Fermentas); 15 μl-20 μM pD_42oligonucleotide (SEQ ID No: 8); 186 μl nuclease-free water and 1.8 μl ofpurified (<1.8*10¹⁰ molecules) TC (translation in Roche—RTS HY kit).Reaction mixture was divided into two tubes 45 μl and 225 μl. To firstpart—45 μl of RT mixture were added 5 μl of nuclease-free water. Thissample was considered to be negative selection control (without dNTP)and has to prove that there is no DNA contamination in the reactionmixture and cDNA synthesis is strictly linked to reverse transcriptasefunctional activity coming from MLV RT in ternary complex. To secondpart—225 μl of RT mixture were added 25 μl-10 mM each dNTP Mix(Fermentas) and reaction mixture again was divided into two tubes—200 μl(4×50 μl) for selection control (<1.2*10¹⁰ molecules of TC totally) and50 μl supplemented with 1 μl-200 u/μl of RevertAid H- M-MuLV ReverseTranscriptase (Fermentas) for positive selection control. According toprotocol each reverse transcription reaction mix contains <3*10⁹molecules of ternary complex in 50 μl volume.

Oil-surfactant mixture for emulsification was prepared by mixing ABIL EM90 (Goldschmidt) into mineral oil (M5904-Sigma) to final concentrationof 4% (v/v) (Ghadessy and Holliger, 2004; U.S.2005/064460). Emulsionswere prepared at +4° C. in 5 ml cryogenic vials (430492-Corning) bymixing 950 μl of oil-surfactant mixture with 50 μl of RT mixture. Mixingwas performed using MS-3000 magnetic stirrer with speed control (Biosan)at ˜2100 rpm; Rotilabo®—(3×8 mm) magnetic followers with centre ring(1489.2-Roth); water phase was added by 10 μl aliquots every 30 sec,continuing mixing for 2 more minutes (total mixing time—4 min).According to optical microscopy data, compartments in our emulsions varyfrom 0.5 μm to 10 μm in size with average diameter of ˜2 μm. Thereforeit was expected to have ˜1*10¹⁰ water in oil compartments after theemulsification of 50 μl reverse transcription reaction mixture, whichcontains less than 3*10⁹ molecules of ternary complex (about 1 mRNA andreverse transcriptase molecules per 3-4 compartments).

All emulsions were incubated one hr at +50° C. in order to select forreverse transcriptase variants, which work better at highertemperatures. To recover the reaction mixtures emulsions were moved to1.5 ml tube, centrifuged for ten min at room temperature and 25,000 g.Oil phase was removed leaving concentrated (but still intact) emulsionat the bottom of the tube. Emulsions were broken by extraction with 0.9ml water-saturated ether; 0.9 ml water-saturated ethyl-acetate (in orderto remove ABIL EM 90 detergent) and again 0.9 ml water-saturated ether.Water phase was dried for five min under vacuum at room temperature and250 μl of PB buffer (Qiagen PCR purification kit) were added. Fourselection samples were merged into two tubes. Synthesized cDNA wasfurther purified with Qiagen PCR purification kit and eluted in 30 μl ofEB buffer (Qiagen PCR purification kit) in case of negative and positiveselection controls and 2×30 μl in case of selection control.

Amplification of cDNA was performed by nested PCR. First of all smallPCR amplification was performed in order to check negative and positiveselection controls and determine minimal number of PCR cycles requiredfor efficient amplification of cDNA in selection samples. PCR mixture(200 μl) was prepared on ice: 20 μl-10× Taq buffer with KCl (Fermentas);20 μl-2 mM of each dNTP (Fermentas); 12 μl-25 mM MgCl₂ (Fermentas); 2.5μl-1 u/μl LC (recombinant) Taq DNA Polymerase (Fermentas); 1 μl-2.5 u/μlPfu DNA Polymerase (Fermentas); 1 μl-100 μM RD_Nde primer (SEQ ID No:9); 1 μl- 100 μM pD_55 primer (SEQ ID No: 10); 50 μl—purified cDNA ofselection controls; 92 μl water. The cycling protocol for 2185 bp PCRfragment was: initial denaturation step was three min at 94° C., 25cycles (45 sec at 94° C., 45 sec at 58° C., and 2 min at 72° C.) andfinal elongation was five min at 72° C.

Nested PCR mixture (500 μl) for full gene amplification was prepared onice: 50 μl—10× Taq buffer with KCl (Fermentas); 50 μl-2 mM of each dNTP(Fermentas); 30 μl-25 mM MgCl₂ (Fermentas); 6.25 μl-1 u/μl LC(recombinant) Taq DNA Polymerase (Fermentas); 2.5 μl-2.5 u/μl Pfu DNAPolymerase (Fermentas); 2.5 μl-100 μM M_Esp primer (SEQ ID No: 13); 2.5μl-100 μM M_Eri primer (SEQ ID No: 14); 50 μl of first PCR (primers setRD_Nde//pD_55); 306 μl water. The cycling protocol for 2077 bp PCRfragment was: initial denaturation step was three min at 94° C., 22cycles (45 sec at 94° C., 45 sec at 55° C., and 2 min at 72° C.) andfinal elongation for five min at 72° C.

Final PCR fragment of selection sample was agarose-gel purified usingQiagen gel extraction kit (elution in 60 μl˜50 ng/μl). Purified PCRfragment was digested with EcoRI and Esp3I for one hr at 37° C. andagain agarose-gel purified (elution in 30 μl˜50 ng/μl).

Recovered MLV reverse transcriptase library after 1^(st) selection roundwas ligated with START and END fragments (construction described earlierin this example) in order to get PCR fragment (FIG. 7) suitable for2^(nd) round of CRD selection (FIG. 6). Ligation reaction (40 μl) wasprepared at room temperature: 4 μl-10× ligation buffer for T4 DNA Ligase(Fermentas); 2 μl-1 u/μl T4 DNA ligase (Fermentas); 4 μl-50 ng/μlselected library digested with Esp3I and EcoRI (˜200 ng or ˜0.9*10¹¹molecules); 1.1 μl-35 ng/μl START fragment digested with NcoI (˜35 ng or˜1.5*10¹¹ molecules); 1.76 μl-35 ng/μl END fragment digested with EcoRI(˜61 ng or ˜1.5*10¹¹ molecules); 27.2 μl—water. Ligation was performedone hr at room temperature.

Ligated MLV RT library was amplified by PCR (300 μl—prepared on ice): 30μl-10× Taq buffer with KCl (Fermentas); 30 μl-2 mM of each dNTP(Fermentas); 18 μl-25 mM MgCl₂ (Fermentas); 24 μl—DMSO (D8418-Sigma);3.7 μl-1 u/μl LC (recombinant) Taq DNA Polymerase (Fermentas); 1.5μl-2.5 u/μl Pfu DNA Polymerase (Fermentas); 1.5 μl-100 μM pro-pIVEXprimer (SEQ ID No: 3); 1.5 μl-100 μM pD-ter-primer (SEQ ID No: 17); 25.5μl—ligated MLV RT library (<0.6*10¹⁰ molecules); 164.3 μl—water. Thecycling protocol for 2702 bp PCR fragment was: initial denaturation step3 min at 94° C., 15 cycles (45 sec at 94° C., 45 sec at 53° C., and 3min at 72° C.) and final elongation 5 min at 72° C. PCR fragment wasagarose-gel purified using Qiagen gel extraction Kit (elution in 30μl˜100 ng/μl).

2^(nd) Selection Round

Second selection round was performed following the general setup of the1^(st) selection round of experimental scheme with minor modificationsin PCR cycles, emulsified RT reaction temperature, and a few moredetails.

All modifications are given below:

-   -   transcription—10 μl-100 ng/μl (˜1000 ng) of agarose-gel purified        PCR fragment were taken; final concentration of mRNA used in        2^(nd) selection round was 1.5 μg/μl;    -   translation—0.8 μl-1.5 μg/μl (˜1.2 μg) of mRNA were taken;    -   emulsified RT reaction was performed 1 hr at 52.5° C.;    -   1^(st) PCR (RD_Nde//pD_55)—24 cycles were performed;    -   2^(nd) (nested) PCR (M_Esp//M_Eri)—23 cycles were performed;    -   final concentration of digested PCR fragment—80 ng/μl;    -   ligation—200 ng (˜0.9*10¹¹ molecules) of MLV RT library were        taken;    -   PCR (on ligation mix)—<0.6*10¹⁰ molecules of selected library        were taken and 15 PCR cycles were performed; concentration of        final agarose-gel purified PCR fragment was 200 ng/μl.

3^(rd) Selection Round

Third selection round was performed following the general setup of1^(st) selection round of experimental scheme with minor modificationsin PCR cycles, emulsified RT reaction temperature, and a few moredetails.

All modifications are given below:

-   -   transcription—5 μl-200 ng/μl (˜1000 ng) of agarose-gel purified        PCR fragment were taken; final concentration of mRNA used in        3^(rd) selection round was 1.5 μg/μl;    -   translation—0.8 μl-1.5 μg/μl (˜1.2 μg) of mRNA were taken;    -   emulsified RT reaction was performed 1 hr at 55° C.;    -   1^(st) PCR (RD_Nde//pD_55)—25 cycles were performed;    -   2^(nd) (nested) PCR (M_Esp//M_Eri)—22 cycles were performed;    -   final concentration of digested PCR fragment—70 ng/μl;    -   ligation—200 ng (˜0.9*10¹¹ molecules) of MLV RT library were        taken;    -   PCR (on ligation mix)—<0.6*10¹⁰ molecules of selected library        were taken and 15 PCR cycles were performed; concentration of        final agarose-gel purified PCR fragment was 100 ng/μl.

4^(th) Selection Round

Fourth selection round was performed following general setup of 1^(st)selection round of the experimental scheme with minor modifications inPCR cycles, emulsified RT reaction temperature, and a few more details.

All modifications are given below:

-   -   transcription—10 μl-100 ng/μl (˜1000 ng) of agarose-gel purified        PCR fragment were taken; final concentration of mRNA used in        4^(th) selection round was 1.8 μg/μl;    -   translation—0.67 μl-1.8 μg/μl (˜1.2 μg) of mRNA were taken;    -   emulsified RT reaction was performed 1 hr at 57.5° C.;    -   1^(st) PCR (RD_Nde//pD_55)—25 cycles were performed;    -   2^(nd) (nested) PCR (M_Esp//M_Eri)—24 cycles were performed;    -   final concentration of digested PCR fragment—50 ng/μl;    -   ligation—200 ng (˜0.9*10¹¹ molecules) of MLV RT library were        taken;    -   PCR (on ligation mix)—<0.6*10¹⁰ molecules of selected library        were taken and 15 PCR cycles were performed; concentration of        final agarose-gel purified PCR fragment was 100 ng/μl.

5^(th) Selection Round

Fifth selection round was performed following general setup of 1^(st)selection round of the experimental scheme with some modifications inPCR cycles, emulsified RT reaction temperature, and the final stage ofanalysis.

All modifications are given below:

-   -   transcription—10 μl-100 ng/μl (˜1000 ng) of agarose-gel purified        PCR fragment were taken; final concentration of mRNA used in        5^(th) selection round was 1.1 μg/μl;    -   translation—1.1 μl-1.1 μg/μl (˜1.2 μg) of mRNA were taken;    -   emulsified RT reaction was performed 1 hr at 60° C.;    -   1^(st) PCR (RD_Nde//pD_55)—25 cycles were performed;    -   2^(nd) (nested) PCR (M_Esp//M_Eri)—33 cycles were performed;

Nested PCR mixture (500 μl) for full gene amplification was prepared onice: 50 μl-10× Taq buffer with KCl (Fermentas); 50 μl-2 mM of each dNTP(Fermentas); 30 μl-25 mM MgCl₂ (Fermentas); 6.25 μl-1 u/μl LC(recombinant) Taq DNA Polymerase (Fermentas); 2.5 μl-2.5 u/μl Pfu DNAPolymerase (Fermentas); 2.5 μl-100 μM M_Esp primer (SEQ ID No: 13); 2.5μl-100 μM M_Hind3+primer (SEQ ID No: 18); 50 μl of first PCR (primersset RD_Nde//pD_55); 306 μl water. The cycling protocol for 2077 bp PCRfragment was: initial denaturation step 3 min at 94° C., 22 cycles (45sec at 94° C., 45 sec at 55° C., and 3 min at 72° C.) and finalelongation 5 min at 72° C.

The final PCR fragment of selection sample was agarose-gel purifiedusing Qiagen gel extraction kit (elution in 60 μl˜60 ng/μl). PurifiedPCR fragment was digested with HindIII and Esp3I for 1 hr at 37° C. andagain agarose-gel purified (elution in 40 μl˜50 ng/μl).

Recovered MLV reverse transcriptase library after 5^(th) selection roundwas ligated into plasmid vector prepared from pET_his_MLV_pD (SEQ ID No:1 and FIG. 2) digested with NcoI and HindIII, giving new 7474 bp plasmidpET_his_MLV (SEQ ID No: 19) encoding MLV RT with N-terminal his-tag andno pD fusion on C-terminus for fast protein purification using affinitychromatography.

Ligated MLV RT library after 5^(th) selection round was electroporatedinto T7 expression strain ER2566. Individual clones were sequenced andanalyzed. Pool of evolved proteins as well as individual mutants andprimary wt M-MuLV reverse transcriptase in the same construction weregrown in 200 ml of LB to A590 ˜0.7 and purified via his-tag by affinitychromatography using 2 ml of Qiagen—Ni-NTA Superflow resin (allpurification was performed under native conditions according tosuppliers recommendations). Elution was performed in 1 ml of EB (50mM—NaH₂PO₄, 300 mM—NaCl, 250 mM—imidazol, pH 8.0, 10mM—β-mercaptoethanol and 0.1% of triton X-100). All proteins weredialyzed against 50 times excess of storage buffer (50 mM Tris-HCl (pH8.3 at 25° C.), 0.1 M NaCl, 1 mM EDTA, 5 mM DTT, 0.1% (v/v) Triton X-100and 50% (v/v) glycerol). Protein purity was checked on SDS-PAGE (usually˜40-80% of target protein). Protein concentration was determined usingBradford method based Bio-Rad protein assay (500-0006).

MLV reverse transcriptase specific activities were measured at 37° C.,50° C. (enzyme diluted in special dilution buffer: 30 mM Tris-HCl pH 8.3at 25° C., 10 mM DTT, 0.5 mg/ml BSA) and rest of activity at 37° C.after five min enzyme incubation at 50° C. (enzyme diluted and itsstability measured in 1×RT reaction buffer: 50 mM Tris-HCl pH 8.3 at 25°C., 4 mM MgCl₂, 10 mM DTT, 50 mM KCl). Enzyme activity in all cases wasassayed in the following final mixture: 50 mM Tris-HCl (pH 8.3 at 25°C.), 6 mM MgCl₂, 10 mM DTT, 40 mM KCl, 0.5 mM dTTP, 0.4 MBq/ml[3H]-dTTP, 0.4 mM polyA•oligo(dT)₁₂₋₁₈. Activity units were determinedmeasuring incorporation of dTMP into a polynucleotide fraction (adsorbedon DE-81) in ten min at particular reaction temperature and comparing toknown amounts of commercial enzyme.

Results

During analysis of selection data we have accumulated 104 sequencesexpressing full length M-MuLV reverse transcriptase. The total CLUSTALWalignment of all proteins (FIG. 17) is compiled without N terminal Histag in order to have the same numeration of amino acids as is usuallyused in literature. Wild type sequence denoted as MLV is always given asthe first sequence. Mutations are marked using white font color in blackbackground (FIG. 17). Amino acids positions, mutations of which somehowimprove M-MuLV reverse transcriptase properties and are described indifferent patent applications are marked in the alignment as columns ofamino acids highlighted in bold font. Mutations originating from ourselection and located in bolded columns serve as proof that ourselection procedure precisely targeted the beneficial hot spot or evenexact amino acid mutations described elsewhere. Out of 104 sequencedclones we found 98 unique sequences and 1 wt (L5_87) sequence. Fivesequences are repeated twice (L5_21 and L5_111; L5_43 and L5_112; L5_49and L5_63; L5_64 and L5_93; L5_85 and L5_96). In total we had randomlyexpressed 55 proteins. Out of 55 expressed proteins 40 enzymaticallyactive mutant variants (including control wt) of M-MuLV reversetranscriptase were successfully purified to 40-80% homogeneity accordingto SDS-PAGE. Total protein concentration in purified RT samples was inrange of 0.6-5.5 mg/ml. Mutant RT variants were tested for reversetranscriptase activity at 37° C., 50° C. and residual activity at 37° C.after 5 min incubation at 50° C. (FIG. 8). Reverse transcriptaseactivity at 37° C. was normalized to be 100% and is omitted in FIG. 8.Thus only two types of columns (percents of RT activity at 50° C. andresidual RT activity at 37° C. after 5 min incubation at 50° C.) areshown. As a control wt M-MuLV reverse transcriptase used for mutantslibrary construction is presented. This primary enzyme was expressed inthe same vector and purified in the same way as mutant variants of RT.An average value of wt enzyme RT activity at 50° C. is about 45%comparing to activity at 37° C. Almost all tested proteins, with fewexceptions, had higher than 45% activity at 50° C. An average value ofRT activity at 50° C. for all tested mutants was about ˜92% and was morethan two times higher comparing to wt enzyme (45%). Some mutants were100% or even more active at 50° C. as they were at 37° C.: 20, 23,L5_16, L5_24, L5_30, L5_35, L5_37, L5_43, L5_46, L5_47, L5_49, L5_52,L5_55, L5_64, L5_65, L5_68, L5_72. The best mutants found had RTactivity at 50° C. about 140% and more (3 times higher than 45% of wt):20 (165%), L5_37 (162%), L5_43 (156%), L5_46 (135%), L5_47 (179%), L5_52(137%), L5_64 (142%) and L5_68 (153%).

Even though majority of mutants had very high RT activities at 50° C.,they were not as thermostable. Residual RT activity at 37° C. after fivemin incubation at 50° C. of wt control was ˜11%. The same averageresidual activity of selected enzymes was also similar ˜12%. Some testedRT variants were substantially more thermostable and had residualactivity 2-3 times higher than wt enzyme (11%): L5_8 (25%), L5_43 (32%),L5_46 (27%), L5_64 (28%), L5_65 (25%), L5_68 (31%).

Specific activity (u/mg of protein) for partially purified wt enzyme was˜200′000 u/mg (FIG. 9). Selected RT variants were expressed and purifiedin very diverse manner and an average specific activity (˜155,000 u/mg)was slightly lower as for wt control (FIG. 9). In some cases specificactivity was decreased, in some—increased (20-˜274′000 u/mg;L5_11-˜273′000 u/mg; L5_28-˜230′000 u/mg; L5_30-˜224′000 u/mg;L5_35-˜316′000 u/mg; L5_43-˜328′000 u/mg; L5_46-˜304′000 u/mg;L5_52-˜310′000 u/mg; L5_64-˜256′000 u/mg; L5_65-˜247′000 u/mg).

The disclosed selection system worked well. Using increased temperatureof RT reaction as a selection pressure factor, we have managed to evolvefaster (specific RT activity at 50° C. is higher) and more thermostable(residual RT activity at 37° C. after five min preincubation at 50° C.)reverse transcriptases.

The source of valuable information was an alignment of selected proteinsequences (FIG. 17). Sequences of analyzed proteins, whose activity at50° C. was substantially better as compared to primary wt M-MuLV (70%and more comparing to 45% of wt activity) are underlined (FIG. 17).Number of mutagenized amino acids varied in range of 0 (wt or L5_87) to12 (L5_9). List of mutations found in all selected RT variants is givenin Appendix 2. Proteins are sorted by decreasing number of mutations.Most mutants (53 out of 104) had 4-6 mutations per sequence. Reversetranscriptase sequence had some hot spots, which were very important andbeneficial for the RT reaction in general and for the thermostability ofenzyme. Those hot spots could be easily identified in multiple sequencealignment (FIG. 17) as a conglomeration of mutations at particularpositions. Especially important were mutations found in betterperforming variants of M-MuLV reverse transcriptase (sequencesunderlined in FIG. 17). Summarized information about the most frequentmutations (in decreasing order) is given in FIG. 18. Mutant proteinswith substantially higher activity at 50° C. are highlighted in black.If the same mutation repeated for many times and tested reversetranscriptases with this mutation were better performing at 50° C., itindicated that this mutation was somehow beneficial for reversetranscription reaction.

According to the frequency with which mutations are found, they can bedivided into five classes: 21-31 repeats; 14-18 repeats; 4-7 repeats;2-3 repeats, and 1 repeat. The first group of the most frequentmutations comprises four amino acids D524 (31 repeats); D200 (30repeats); D653 (23 repeats) and D583 (21 repeats). Two amino acids (D524and D583) are known to complex magnesium ions in active centre ofribonuclease H domain. Mutants D524G, D583N and E562Q are used to turnoff RNase H activity of M-MuLV reverse transcriptase (Gerard et al.,2002), what improves synthesis of cDNA. Results of our selection werestrikingly similar. Mutation of aspartate 524 was found in 31 sequencesout of 98. Moreover, D524N substitution was found once, D524A—10 timesand finally D524G—20 times. Thus our selection not only preciselytargeted important amino acids, but also the same amino acidsubstitutions, which are known to be the best. Exactly the samesituation was with mutation of aspartate 583, which was repeated in 21selected proteins out of 104. Substitution D583E was found once, D583A—3times, D583G—7 times and finally D583N—10 times. Again, the same aminoacid and same substitution (D583N), which was known to be the best, wasselected most frequently. Commercial enzyme SUPERSCRIPT II fromInvitrogen has three mutations: D524G, D583N and E562Q (WO2004024749).It is of interest that mutation of the third amino acid substitutionE562 in our selection was found only once (E562K in L5_71). Thissuggested that most likely glutamate 562 was not as important asaspartates 524 and 583, or for some reasons exchange of this amino acidcan cause some side effects and is not beneficial for RT reactionsperformed at increased temperatures (>50° C.).

Further analysis of selected proteins sequences allowed identificationof many more hot amino acids positions, mutations of which are describedfor improved M-MuLV reverse transcriptase: H204R—7 repeats (U.S. Pat.No. 7,078,208); H638R—4 repeats (U.S. 2005/0232934A1); T197A—2 repeats(U.S. Pat. No. 7,056,716); M289V(L), T306A(M)—2 repeats (U.S. Pat. No.7,078,208); E302K, N454K—2 repeats (WO 07022045A2); E69G, L435P—1sequence (WO 07022045A2); Y64C, Q190R, V223M, F309S—1 sequence (U.S.Pat. No. 7,056,716); E562K—1 sequence (U.S. Pat. No. 7,078,208). Therewere also two selected sequences of reverse transcriptases which hadcombination of three amino acids substitutions described in theliterature (30—D200N, T306M, D524N, D583G; L5_28—T306A, F309S, D524A,H594R, F625S).

In addition to known mutations, we have identified many more amino acidspositions which are mutated quite often: D200N(A, G)—30 repeats;D653N(G, A, H, V)—23 repeats; L603W(M)—18 repeats; T330P—15 repeats;L139P—14 repeats; Q221R—6 repeats; T287A—6 repeats; I49V(T)—5 repeats;N479D—5 repeats; H594R(Q)—5 repeats; F625S(L)—5 repeats; P65S—4 repeats;H126S(R)—4 repeats; L333Q(P)—4 repeats; A502V—4 repeats; E607K(G, A)—4repeats; K658R(Q)—4 repeats; H8P(R)—3 repeats; P130S—3 repeats; E233K—3repeats; Q237R—3 repeats; N249D—3 repeats; A283D(T)—3 repeats; A307V—3repeats; Y344H—3 repeats; P407S(L)—3 repeats; M428L—3 repeats; Q430R—3repeats; D449G(A)—3 repeats; A644V(T)—3 repeats; N649S—3 repeats;L671P—3 repeats; E673G(K)—3 repeats; N678I—3 repeats (FIG. 18).

Best performing variants of RT usually have mutations of amino acids,which are modified most frequently:

20 (50° C.-123%)-—200N (30 repeats), L603W (18 repeats) and slightlymodified C terminus—N678I, S679P, R680A;L5_35 (50° C.-125%)—D200N (30 repeats), T330P (15 repeats), N479D (5repeats);L5_37 (50° C.-162%)—H123S (4 repeats), L149F (1 sequence), D200N (30repeats), N454K (2 repeats), D583N (21 repeats);L5_43 (50° C.-160%)—D200N (30 repeats), Q237R (3 repeats), T330P (15repeats), D524G (31 repeats), F625S (5 repeats), D653N (23 repeats);L5_46 (50° C.-135%)—D200N (30 repeats), T330P (15 repeats), D583N (21repeats), T644T (3 repeats);L5_47 (50° C.-179%)—N107S (1 repeat), H126R (4 repeats), T128A (1repeat), I179V (2 repeats), D200N (30 repeats), H642Y (2 repeats), D653N(23 repeats);L5_52 (50° C.-137%)—D200N (30 repeats), T330P (15 repeats), Q374R (2repeats), (D583N (21 repeats);L5_64 (50° C.-142%)—D200N (30 repeats), D216G (2 repeats), D524G (31repeats), E545G (2 repeats);L5_65 (50° C.-127%)—D200N (30 repeats), Q238H (1 repeat), L570I (1repeat), L603W (18 repeats);L5_68 (50° C.-153%)—M39V (2 repeats), I49V (2 repeats), Q91R (2repeats), H204R (7 repeats), T287A (6 repeats), N454K (2 repeats), F625L(5 repeats), D653H (23 repeats).

The combined data set of measured RT activities and sequence alignmentanalysis of mutant proteins allowed us to determine many beneficialmutations and combinations thereof in the sequence of M-MuLV reversetranscriptase sequence.

EXAMPLE 3 Analysis of Moloney Murine Leukemia Virus ReverseTranscriptase Mutants

The in vitro evolution experiment described in Example 2 was veryefficient. Gradually increased temperature of reverse transcriptionreaction was used as a selection pressure and generated many differentmutant variants of M-MuLV RT. Most were able to perform better atelevated temperatures compared to primary enzyme. Sequence analysis ofevolved reverse transcriptases indicates hot spots and most importantamino acids positions (replacements), responsible for compleximprovement of enzyme properties. In order to elucidate individualimpact of different mutations single and multiple mutants of M-MuLV RTwere constructed, partially purified and analyzed. Starting point formutants construction was 7474 bp plasmid pET_his_MLV (SEQ ID No: 19)encoding M-MuLV RT with N-terminal his-tag for fast protein purificationusing affinity chromatography. M-MuLV reverse transcriptase specificactivity at 37° C., relative activity at 50° C. and relative residualactivity at 37° C. after five min enzyme incubation at 50° C. weredetermined. In some cases RNase H activity was checked and cDNAsynthesis reaction at different temperatures on 1 kb or 4.5 kb RNA wasperformed.

Methods and Materials

Initial plasmid pET_his_MLV (SEQ ID No: 19) was used as a startingmaterial for mutagenic PCR. Mutations were introduced using mutagenicprimers. Individual clones were sequenced and analyzed. M-MuLV RTmutants were expressed in T7 expression strain ER2566. Individualproteins and primary wt M-MuLV reverse transcriptase in the sameconstruction were grown in 200 ml of LB to A590 ˜0.7 and purified viahis-tag by affinity chromatography using 2 ml of Qiagen—Ni-NTA Superflowresin (all purification was performed under native conditions accordingto suppliers recommendations). Elution was performed in 1 ml of EB (50mM—NaH₂PO₄, 300 mM—NaCl, 250 mM—imidazol, pH 8.0, 10mM—β-mercaptoethanol and 0.1% of triton X-100). All proteins weredialyzed against 50 times excess of storage buffer (50 mM Tris-HCl (pH8.3 at 25° C.), 0.1 M NaCl, 1 mM EDTA, 5 mM DTT, 0.1% (v/v) Triton X-100and 50% (v/v) glycerol). Protein purity was checked on SDS-PAGE (usually˜40-80% of target protein). Protein concentration was determined usingBradford reagent (Fermentas #R1271).

MLV reverse transcriptase activities were measured at 37° C., 50° C.(enzyme diluted in special dilution buffer: 30 mM Tris-HCl pH 8.3 at 25°C., 10 mM DTT, 0.5 mg/ml BSA) and rest of activity at 37° C. after fivemin enzyme incubation at 50° C. (enzyme diluted and its stabilitymeasured in 1×RT reaction buffer: 50 mM Tris-HCl pH 8.3 at 25° C., 4 mMMgCl₂, 10 mM DTT, 50 mM KCl). Enzyme activity in all cases was assayedin the following final mixture: 50 mM Tris-HCl (pH 8.3 at 25° C.), 6 mMMgCl₂, 10 mM DTT, 40 mM KCl, 0.5 mM dTTP, 0.4 MBq/ml [3H]-dTTP, 0.4 mMpolyA•oligo(dT)₁₂₋₁₈. Activity units were determined measuringincorporation of dTMP into a polynucleotide fraction (adsorbed on DE-81)in ten min at particular reaction temperature and comparing to knownamounts of commercial enzyme. RNase H activity of M-MuLV reversetranscriptase variants was measured according to U.S. Pat. No.5,405,776. RNase H activity of purified enzymes was assayed in reactionmixtures (50 μl) containing 50 mM Tris-HCl pH 8.3, 2 mM MnCl₂, 1 mM DTTand [3H](A)n*(dT)n (5 μM [3H](A)n, 35 cpm/pmol; 20 μM (dT)n). Reactionswere incubated at 37° C. for 10 min and were stopped by adding 10 μl oftRNA (1 mg/ml) and 20 μl of cold 50% TCA. After ten min on ice, themixture was centrifuged for ten min in an Eppendorf centrifuge (at 25000g). Forty μl of supernatant was counted in a LSC-universal cocktail(Roth-Rotiszint eco plus). One unit of RNase H activity is the amount ofenzyme required to solubilize one mole of [3H](A)n in [3H](A)n*(dT)n in10 min at 37° C.

“RevertAid™ First Strand cDNA Synthesis Kit” (#K1622-Fermentas) and itscontrol 1.1 kb RNA with a 3′-poly(A) tail in combination witholigo(dT)₁₈ primer was used to check purified reverse transcriptases fortheir ability to synthesize cDNA at different temperatures.Alternatively 4.5 kb RNA (synthesized from Eco31I linearized pTZ19Rplasmid, which additionally contains piece of phage lambda DNA 5505-8469bp) was used to test reverse transcription reaction. cDNA wassynthesized one hr in 20 μl reaction volume using kit's components 1 μgof synthetic RNA, following provided protocol with only minormodifications (without 5 min preincubation at 37° C.). Reversetranscription reactions were performed in 96 well PCR plate in EppendorfMastercycler gradient PCR machine applying corresponding temperaturegradient. Synthesized cDNA was analyzed by alkaline agarose gelelectrophoresis (staining with ethidium bromide). Samples of cDNAsynthesis analysis on alkaline agarose gels are given in FIG. 16.

Results

According to the frequency with which mutations are found during theevolution of M-MuLV reverse transcriptase, they can be divided into fiveclasses: 21-31 repeats; 14-18 repeats; 4-7 repeats; 2-3 repeats and 1repeat. Construction of individual reverse transcriptase mutants ingeneral was performed according to this information. Most frequentlyfound mutations were tested first. Reverse transcriptase specificactivity at 37° C., relative activity at 50° C. and relative residualactivity at 37° C. after 5 min enzyme incubation at 50° C. weredetermined. In some cases RNase H activity was checked and cDNAsynthesis reaction at different temperatures on 1 kb or 4.5 kb RNA wasperformed. All experimental data on individual mutants are presented inFIG. 19. Second column (“selection frequency”) indicates the number ofsequenced mutants, which had exact mutation and the number in theparentheses indicates total number of particular amino acid mutationsfound in selection. For example D200N—25 (30) means that aspartate 200replacement to asparagine was found 25 times out of 30 total D200mutations. Reverse transcriptase specific activity measured at 37° C.was given in units per mg of protein. Relative enzyme activity at 50° C.and relative residual RT activity at 37° C. after five min incubation at50° C. was given in percent normalized to specific activity (100%) ofthe same enzyme measured at 37° C. As a control (first line) is given wtM-MuLV reverse transcriptase used for mutants library construction. Thisprimary enzyme was expressed in the same vector and purified in the sameway as mutant variants of RT. Specific activity of wt enzyme was about200,000 u/mg at 37° C., relative activity at 50° C. (comparing toactivity at 37° C.)—45-50% (90,000-100,000 u/mg) and relative residualRT activity at 37° C. after five min incubation at 50° C. (comparing toactivity at 37° C.) was about 11% (˜22,000 u/mg). Wild type enzyme hasabout 160-200 u/mol of RNase H activity and can synthesize full length 1kb cDNA at 48° C. It is known that M-MuLV reverse transcriptase isprotected from thermal inactivation by the binding to template-primersubstrate and contrary, enzyme is less thermostable in solution alone(Gerard et al., 2002). Relative residual RT activity at 37° C. afterfive min incubation at 50° C. directly indicated enzyme thermostabilityin solution without substrate. Meanwhile relative activity at 50° C.represented enzyme thermostability in complex with RNA/DNA substrate andspeed of cDNA synthesis. Fast mutant variant of reverse transcriptasegives increased numbers of polymerase units at 50° C., even if itsthermostability will be the same as wild type enzyme. Highesttemperature of cDNA synthesis, in our case 1 kb or 4.5 kb is the mostcomprehensive parameter, which represents general ability of enzyme tosynthesize cDNA at increased temperatures. Reverse transcriptasemutants, which have at least 10% increased: specific activity at 37°(≥220′000 u/mg, 200′000 u/mg-wt), relative activity at 50° C. comparingto mutant activity at 37° C. (≥54%, 45-50%-wt), or relative residualactivity at 37° C. after five min incubation at 50° C. comparing tomutant activity at 37° C. (≥13%, 11%-wt),—are shadowed in black andconsidered as significantly improved enzymes. Mutants able to synthesizefull length 1 kb cDNA at temperatures higher than 48° C. are alsoshadowed in black.

Reverse transcriptase mutants with increased specific activity (≥220′000u/mg) at 37° C. were (FIG. 19):

D200 (D200N—254′000 u/mg; D200G—276′000 u/mg; D200H—234′000 u/mg),T330 (T330N—223′000 u/mg; T330D—240′000 u/mg),Q221 (Q221R—268′000 u/mg),H594 (H594K—270′000 u/mg; H594Q—231′000 u/mg),D449 (D449E—224′000 u/mg; D449N—221′000 u/mg),M39 (M39N—349′000 u/mg),M66 (M66L—237′000 u/mg; M66V—227′000 u/mg; M66I—240′000 u/mg),H126 (H126R—227′000 u/mg),W388 (W388R—266′000 u/mg),I179 (I179V—251′000 u/mg).

Reverse transcriptase mutants with increased relative activity (≥54%) at50° C. (comparing to activity at 37° C.) were (FIG. 19):

D200 (D200N—84%; D200A—87%; D200Q—103%; D200E—79%; D200V—131%;D200W—103%; D200G—88%; D200K—102%; D200R—68%; D200H—54%), L603(L603W—105%; L603F—104%; L603Y—95%; L603M—77%), D653 (D653N—93%;D653K—106%; D653A—99%; D653V—98%; D653Q—93%; D653L—83%; D653H—116%;D653G—90%; D653W—93%; D653E—80%), T330 (T330P—80%; T330N—69%; T330D—55%;T330V—65%; T330S—67%), Q221 (Q221R—94%; Q221K—77%; Q221E—64%; Q221M—58%;Q221Y—77%), E607 (E607K—84%; E607A—98%; E607G—72%; E607D—69%), L139(L139P—59%), T287 (T287S—68%), N479 (N479D—81%), H594 (H594R—69%;H594K—80%; H594Q—75%; H594N—61%), D449 (D449G—79%; D449E—77%; D449N—75%;D449A—99%; D449V—83%), M39 (M39V—54%; M39N—71%), M66 (M66L—79%;M66V—73%; M66I—80%), L333 (L333Q—54%), H126 (H126R—58%), P130(P130S—70%), Q91 (Q91—56%),

W388 (W388R—72%),

R390 (R390W—64%), Q374 (Q374R—56%), E5 (E5K—67%).

Reverse transcriptase mutants with increased relative residual activity(≥13%) at 37° C. after five min incubation at 50° C. (comparing toactivity at 37° C.) were (FIG. 19):

D200 (D200N—15%; D200A—18%; D200Q—23%; D200R—27%; D200H—27%), L603(L603W—23%; L603Y—13%; L603P—15%), D653 (D653N—21%; D653K—15%;D653A—18%; D653V—16%; D653Q—18%; D653H—13%; D653G—13%; D653W—13%;D653E—19%), T330 (T330P—21%; T330N—13%; T330D—16%; T330S—15%), T287(T287A—13%; T287F—13%), H594 (H594R—14%; H594Q—13%), D449 (D449G—13%),M39 (M39V—13%), M66 (M66L—13%), Y344 (Y344H—13%), Q91 (Q91R—13%), N649(N649S—16%), W388 (W388R—14%).

Mutants able to synthesize full length 1 kb cDNA at temperatures higherthan 48° C. were (FIG. 19):

D200 (D200N—50.4° C.; D200H—50.4° C.), L603 (L603W—53.1° C.; L603F—50.4°C.; L603Y—47.8-50.4° C.),

D653 (D653N—50.4-53.1° C.; D653K—50.4-53.1° C.; D653A—50.4° C.;D653V—50.4° C.; D653Q—50.4° C.; D653L—50.4° C.; D653H—50.4-53.1° C.;D653G—50.4° C.; D653W—50.4° C.),

Q221 (Q221R—50.4° C.), E607 (E607K—47.8-50.4° C.), H594(H594K—47.8-50.4° C.; H594Q—47.8-50.4° C.).

Samples of 1 kb cDNA synthesis analysis on alkaline agarose gels aregiven in FIG. 16 A-D.

According to collected biochemical data, most important positions inM-MuLV reverse transcriptase sequence, which can impact cDNA synthesisat increased temperatures, are: D200, L603, D653, T330, Q221, E607,L139, T287, N479, H594, D449, M39, M66, L333, H126, Y344, P130, Q91,N649, W388, R390, I179, Q374, E5.

In general mutations of interest can be combined inbetween and M-MuLVreverse transcriptase thermostability with and without substrate,velocity, processivity and overall ability to synthesize cDNA atincreased temperatures can be further improved. Some data, whichillustrates enzyme improvement by combinatorial approach, are presentedin FIG. 20. Single mutants D200N and L603W had relative activity at 50°C. 84% and 105%. Highest temperatures of 1 kb cDNA synthesis was 50.4°C. and 53.1° C. Double mutant D200N; L603W had relative activity 131% at50° C. and can synthesize 1 kb cDNA at 56° C. Triple mutant D200N;L603W; T330P (80% at 50° C.; 1 kb cDNA at 47.8° C.) was improved furtherand had relative activity 175% at 50° C. and could synthesize 1 kb cDNAat 56-58° C. Quadruple mutant D200N; L603W; T330P; E607K (84% at 50° C.;1 kb cDNA at 47.8-50.1° C.) had relative activity 174% at 50° C. andcould synthesize 1 kb cDNA at 60-62° C. Quintuple mutant D200N; L603W;T330P; E607K; L139P (59% at 50° C.; 1 kb cDNA at 47.8° C.) had relativeactivity 176% at 50° C. and could synthesize 1 kb cDNA at 62° C. andthat was about 14° C. higher temperature compared to wild type M-MuLVreverse transcriptase (1 kb cDNA at 47.8° C.). Additive character ofthermostability was also observed in case of: N479D, H594R mutants(D200N; L603W—131% at 50° C., 1 kb cDNA at 56° C. versus D200N; L603W;N479D; H594R—182% at 50° C., 1 kb cDNA at 56-58° C., 4.5 kb cDNA at56-58° C.),

T330P mutant (D200N; L603W; D653N; D524G—155% at 50° C., 1 kb cDNA at58-60° C. versus D200N; L603W; D653N; D524G; T330P—180% at 50° C., 1 kbcDNA at 60-62° C.).

Samples of 4.5 kb cDNA synthesis analysis on alkaline agarose gels aregiven in FIG. 16 E-G.

EXAMPLE 4 Modification of CRD—Selection for DNA Dependent DNA PolymeraseActivity Using Biotin-dUTP (Proof of Principle)

This example illustrates activity-based selection strategy of reversetranscriptase as DNA dependent DNA polymerase, which was able toincorporate modified nucleotides into DNA-DNA substrate. The principlescheme of selection is schematically illustrated in FIG. 12. Twoplasmids pET_his_MLV_D583N_pD (encoding RNase H minus Moloney MurineLeukemia Virus (M-MLV) reverse transcriptase fused to protein D spacer)and its derivative pET_his_del_pD (encoding inactivated reversetranscriptase; 57 amino acids deletion in pol domain and mutation D583Nin RNase H domain, Example 1, SEQ ID No: 2) were used as a startingmaterial in this example. Initial DNA fragments encoding active andinactive reverse transcriptases were synthesized in two separatepolymerase chain reactions using plasmids pET_his_MLV_D583N_pD andpET_his_del_pD as a target. Synthesized PCR fragments were used intranscription reaction for synthesis of mRNA, which lacks STOP codon atthe 3′ end. Purified mRNAs were mixed to a ratio of 1:20=MLV (activeRT):del (inactive RT). Double stranded DNA adapter (required forselection of DNA dependent DNA polymerase activity) was ligated to 3′mRNA by T4 DNA ligase. This mRNA-dsDNA complex is used for in vitrotranslation reaction. The ribosome moving along the mRNA stopstranslation at the site of RNA-DNA hybridization (Tabuchi et al. 2001).Ribosome-mRNA/dsDNA-protein complexes were stabilized (as inconventional ribosome display) by dilution of translation mixture withice-cold buffer containing 50 mM Mg²⁺. Mixture of ternary complexes (TC)was purified by ultracentrifugation on sucrose cushions. Purifiedternary complexes (<3*10⁹ molecules taken) containing mRNA-dsDNA linkedto in vitro translated M-MuLV (RNase H-) reverse transcriptases wereused to prepare reaction mixture additionally supplemented withbiotin-dUTP and reaction buffer. Ice-cold reaction mixture wasemulsified yielding ˜1*10¹⁰ water in oil compartments ˜2 μm in size.Emulsified reaction mixture (less than one TC,ribosome-mRNA/dsDNA-protein per compartment) was incubated for 30 min at37° C. After the temperature of compartmentalized reaction mixture wasraised most TCs dissociated releasing mRNA/dsDNA and reversetranscriptase. Successful incorporation reaction could occur only incompartments containing active M-MuLV (RNase H-) reverse transcriptaseresulting in biotinylation of mRNA/dsDNA complex. Biotinylated complexcan be selectively immobilized on streptavidin coated magnetic beads andspecifically amplified by RT-PCR. As a result of successful experimentgenes encoding active enzyme (in our case RT-PCR fragment ofMLV_D583N_pD reverse transcriptase) should be enriched over genesencoding inactive enzyme (del_pD).

Methods and Materials Production of mRNA/dsDNA Complex

(1) Determination of ligation efficiency.

Efficiency of ligation reaction was determined by ligation of MLV_pDmRNA (synthesized from pET_his_MLV_pD plasmid, Example 1) with primerLong+Tb (SEQ ID No: 23) using ddC-Long2 primer (SEQ ID No: 22) as asplint. The 5′ end of Long+Tb was previously phosphorylated using T4Polynucleotide Kinase (Fermentas).

Annealing mix of 36 μl was prepared by mixing of 8.5 pmol (˜10 μg) ofpurified MLV_pD mRNA with four times molar excess of Long+Tb (SEQ ID No:23) and 4.2 times of molar excess of ddC-Long2 (SEQ ID No: 22) innuclease free water. The mixture was incubated at 70° C. for five minand then cooled to room temperature for 20 min. Before adding ligationreaction components mixture was moved to cooling stand for two min.

4.5 μl of 10× ligation buffer and 4.5 μl of T4 DNA ligase (5 v/μl)(Fermentas) were added to 36 μl of annealing mixture. Ligation reactionwas performed for 30 min at 37° C. and followed extraction once withequal volume of Roti® Phenol/chloroform (ROTH) and twice with equalvolume of chloroform. Assuming that mRNA amount was close to initialamount taken for ligation reaction ˜5 μg ligation products mix wasdiluted to 43 μl by nuclease free water. Aliquot of 2 μl of resultantmixture was left for analysis on agarose gel before immobilization onDynabeads M-280 Streptavidin beads (Dynabeads® kilobase BINDER™ Kit(DYNAL Biotech)).

Ten μl resuspended Dynabeads was transferred to a 1.5 ml microcentrifugetube and washed with 50 μl Binding Solution provided in the kit. Thetube was placed on a magnet for 1-2 min until beads settled at the tubeand solution was removed. Dynabeads were gently resuspended by pipetting38 μl of Binding Solution supplemented with 2 μl aqueous tRNA (tRNA fromyeast (Roche)) solution (1 μg/μl) to minimize non-specific mRNA binding.40 μl of Dynabeads in Binding solution was added to solution (˜40 μl)containing ligation products mix. The tube was incubated shaking at +22°C. in termomixer (Eppendorf) for 60 min. After binding of ligatedmRNA/dsDNA, supernatant was removed and the beads were washed threetimes with 50 μl Washing Solution (supplied in the kit). CollectedDynabeads with immobilized ligated mRNA/dsDNA complex was resuspended in26 μl nuclease free water and extracted once with 40 μl Roti®Phenol/chloroform (ROTH) and twice with 40 μl volume chloroform torelease mRNA/dsDNA complex from magnetic beads. One, 2 m and 5 μl offinal mix were analyzed on an agarose gel along with sample (2 μl) leftbefore immobilization and Mass Ruler™High Range DNA ladder. The amountof mRNA in mRNA/DNA complex purified on streptavidin beads wasdetermined comparing mRNA amounts before and after immobilization. Theyield of recovered mRNA was ˜60%, meaning that at least 60% of mRNA wassuccessfully ligated with DNA duplex, resulting in mRNA/dsDNA complex.

(2) Determination of biotin-dUTP incorporation efficiencies intomRNA/dsDNA complex and into self primed mRNA.

As shown in first mRNA to dsDNA ligation experiment, ligation reactionefficiency was ˜60% and higher. Free mRNA left in the ligation mixturecan self prime and participate in extension reaction using M-MuLVreverse transcriptase and biotin-dUTP. This experiment was performed todemonstrate that mRNA/dsDNA complex (ligation product) was a bettersubstrate than free self primed mRNA.

MLV_D583N_pD mRNA was ligated with long+ oligonucleotide (SEQ ID No: 21)according to the procedure described above (construction of initialplasmids pET_his_MLV_D583N_pD and pET_his_del_pD in details as describedin Example 1). About 12.5 ng prepared (MLV_D583N_pD mRNA/long+)substrate was combined with ˜12.5 ng of del_pD mRNA previously incubatedat 70° C. for five min, then cooled to room temperature for 20 min innuclease free water, in a total volume of 12.5 μl. The second mix fordTTP or biotin-dUTP incorporation by reverse transcriptase was prepared:8 μl of 5× reaction buffer for reverse transcriptase; 1 μl-40 u/μlRiboLock™ RNase inhibitor (Fermentas); 18.6 μl nuclease-free water; 0.4μl-200 u/μl RevertAid™ Minus M-MuLV Reverse transcriptase (Fermentas).The prepared mix was divided to two tubes 2×15 μl and 1 μl of 1 mM dTTP(Fermentas) or 1 μl of 1 mM biotin-dUTP (Fermentas) was added.Subsequentially 5 μl substrate (from the first mix) was added to dTTPand biotin-dUTP containing mixtures. Reactions were carried out at 37°C. for 60 min. After that, 1 μl 0.5M EDTA (pH 8.0) was added to bothsamples and reaction mixtures were extracted once with equal volume ofRoti® Phenol/chloroform (ROTH) and once with equal volume of chloroformfollowed by purification on G-50 MicroColumns (GE Healthcare). Two μl ofresultant reaction products were left for direct RT reaction (withoutstreptavidin beads purification), and the remaining part of solutionswere used for biotinylated mRNA-dsDNA complex immobilization onDynabeads M-280 Streptavidin beads (Dynabeads® kilobase BINDER™ Kit(DYNAL Biotech)). Ten μl of resuspended Dynabeads was transferred to a1.5 ml microcentrifuge tube, washed with 25 μl of provided BindingSolution. The tube was placed on the magnet for 1-2 min until beadssettled at the bottom of the tube and the solution was removed.Dynabeads were gently resuspended by pipetting in 90 μl BindingSolution, and 2 μl aqueous tRNA (tRNA from yeast (Roche)) solution(1μg/μl) was added to minimize non-specific mRNA binding. Forty μlDynabeads in Binding Solution was added to solution (˜40 μl) containingthe elongated by dTTP and biotin-dUTP RNA-DNA fragments. The tubes wereincubated at +22° C. in termomixer (Eppendorf) for 40 min by shaking(1400 rpm). After the binding step, supernatant was removed and thebeads were washed three times with 50 μl Washing Solution (provided inthe kit), shaking (1400 rpm) for five min at +22° C. Collected Dynabeadswith immobilizes elongated mRNA-dsDNA complex was resuspended in reversetranscription reaction mixture. Reverse transcription reaction mixturewas prepared on ice: 20 μl-5× reaction buffer for Reverse Transcriptase(Fermentas); 10 μl-10 mM dNTP (Fermentas); 2.5 μl-40 u/μl RiboLock RNaseinhibitor (Fermentas); 5 μl-20 μM pD_42 oligonucleotide (SEQ ID No: 8);2.5 μl RevertAid™ Minus M-MuLV Reverse transcriptase (200u/μl)(Fermentas); 55 μl nuclease-free water. Prepared mix was dividedinto five 19 μl (5×19 μl) aliquots: two were used to resuspend Dynabeadswith immobilized elongated mRNA/dsDNA complex or mRNA, the other twowere transferred to the tubes with samples left without streptavidinbeads purification, and to the rest of the 19 μl aliquot was added to 1μl of nuclease free water—negative reaction control to prove that thereaction mixture was not contaminated by DNA. All reaction mixtures wereincubated by shaking (1000 rpm) at +42° C. in termomixer (Eppendorf) forone hour until cDNA synthesis reaction was complete.

Amplification of cDNA was performed by nested PCR. The initial PCRmixture was prepared on ice: 14 μl-10× Taq buffer with KCl (Fermentas);14 μl-2 mM of each dNTP (Fermentas); 8.4 μl-25 mM MgCl₂ (Fermentas); 2.8μl-1 u/μl LC (recombinant) Taq DNA Polymerase (Fermentas); 0.7 μl-100 μMM_F primer (SEQ ID No: 11); 0.7 μl-100 μM M_2R primer (SEQ ID No: 12);92.4 μl water—mixture was divided into 7 samples for 19 μl (7×19 μl). To5×19 μl of PCR master mix were added 1 μl of cDNA (1-5 RT samples); tothe 6^(th) tube of PCR master mix 1 μl of water was added—negative PCRcontrol; to the 7^(th) tube (positive PCR control)—1 μl ofpET_his_MLV_D583N_pD plasmid (˜1 ng) was added. The cycling protocolwas: initial denaturation step 3 min at 94° C., 25 cycles (45 sec at 94°C., 45 sec at 57° C., and one min at 72° C.) and final elongation forthree min at 72° C.

Predicted amplicons size 907 bp for MLV_D583N_pD and 736 bp for del_pDcDNA. PCR products were analyzed on 1% agarose gel loading 10 μl of PCRmix per well (FIG. 13).

As expected, efficient cDNA amplification was observed only inelongation reaction with biotin-dUTP. Very faint bands of amplified cDNAcould be detected in elongation reaction with dTTP and could beexplained by weak nonspecific binding of mRNA to streptavidin beads.After purification on streptavidin beads DNA encoding MLV_D583N_pD gene(907 bp) was enriched over DNA of del_pD (736 bp). That means mRNA/dsDNAcomplex was elongated by biotin-dUTP much more efficiently than selfprimed del_pD mRNA.

(3) Preparation of mRNA mixture (MLV_D583N_pD:del_pD=1:20) and RNA/dsDNAcomplex.

Preparation of PCR fragments for in vitro transcription. PCR mixture wasprepared on ice: 20 μl-10× Taq buffer with KCl (Fermentas); 20 μl-2 mMof each dNTP (Fermentas); 12 μl-25 mM MgCl₂ (Fermentas); 16 μl—DMSO(D8418-Sigma); 4 μl-1 u/μl LC (recombinant) Taq DNA Polymerase(Fermentas); 1 μl-100 μM pro-pIVEX primer (SEQ ID No: 3); 1 μl-100 μMpD-ter-primer (SEQ ID No: 20); 122 μl water—mixture divided into twotubes 2×98 μl. To 2×98 μl of PCR master mix were added either 2 μl ofpET_his_MLV_D583N_pD (diluted to ˜1 ng/μl) or 2 μl of pET_his_del_pD(diluted to ˜1 ng/μl) (construction of initial plasmidspET_his_MLV_D583N_pD and pET_his_del_pD in details as described inExample 1). The cycling protocol was: initial denaturation step threemin at 94° C., 30 cycles (45 sec at 94° C., 45 sec at 53° C., and twomin at 72° C.) and final elongation for five min at 72° C. Amplificationefficiency was ˜7000 fold from 2 ng of plasmid (7873 bp) target to ˜5 μg(50 ng/μl) of amplified product (2702 bp PCR fragment MLV_D583N_pD frompET_his_MLV_D583N_pD; 2531 bp PCR fragment del_pD from pET_his_del_pD).

Transcription mixture was prepared: 80 μl-5× T7 transcription buffer (1M HEPES-KOH pH 7.6; 150 mM Mg acetate; 10 mM spermidine; 0.2 M DTT); 56μl-112 mM of each NTP (Fermentas); 16 μl- 20 u/μl T7 RNA polymerase(Fermentas); 8 μl-40 u/μl RiboLock RNase inhibitor (Fermentas); 114 μlnuclease-free water—mixture divided into two tubes 2×165 μl and add 35μl-20 ng/μl of PCR fragment MLV_D583N_pD (unpurified PCR mixture) or 35μl-20 ng/μl of PCR fragment del_pD PCR (unpurified PCR mixture).Transcription was performed two hr at 37° C.

Both transcription mixtures were diluted to 200 μl with ice-coldnuclease-free water and 200 μl of 6 M LiCl solution were added. Mixtureswere incubated 25 min at +4° C. and centrifuged for 25 min at +4° C. incooling centrifuge at max speed (25,000 g). Supernatant was discardedand RNA pellet washed with 500 μl of ice-cold 75% ethanol. Tubes werecentrifuged again for five min at +4° C. at max speed and supernatantwas discarded. RNA pellet was dried for five min at room temperature andsubsequently resuspended in 400 μl of nuclease-free ice-cold water byshaking for 15 min at +4° C. and 1400 rpm. Tubes were centrifuged againfor five min at +4° C. at max speed to separate undissolved RNA. About380 μl supernatant was transferred to a new tube with 42 μl 10× DNase Ibuffer (Mg²⁺) (Fermentas); 3 μl -1 u/μl DNaseI (RNase-free) (Fermentas)and incubated for 20 min at +37° C. to degrade DNA. Reaction mixtureswere extracted once with equal volume of Roti® Phenol/chloroform (ROTH)and twice with equal volume of chloroform to remove DNaseI. Forty-threeμl 3 M sodium acetate pH 5.0 solution and 1075 μl of ice-cold 96%ethanol were added to each tube. Finally RNA was precipitated byincubation for 30 min at −20° C. and centrifugation for 25 min at +4° C.at max speed (25,000 g). Supernatant was discarded and RNA pellet washedwith 500 μl of ice-cold 75% ethanol. Tubes were centrifuged again for 4min at +4° C. at max speed and supernatant was discarded. RNA pellet wasdried for five min at room temperature and subsequently resuspended in150 μl nuclease-free ice-cold water by shaking (1400 rpm) for 15 min at+4° C. RNA solution was aliquot for 10 μl and liquid nitrogen frozen.Concentration of mRNA was measured spectrophotometrically anddouble-checked on agarose gel along with RiboRuler™ RNA Ladder, HighRange (Fermentas).

mRNA/dsDNA complex was produced by ligation of long+oligodeoxynucleotide to mRNA using ddC-Long2 oligodeoxynucleotide as asplint. The 5′ end of long+ was previously phosphorylated using T4Polynucleotide Kinase (Fermentas). Oligodeoxynucleotide ddC-Long2 has 3′end modification (ddC) in order to prevent possibility of 3′ endextension by reverse transcriptase on its natural RNA-DNA substrate.

Annealing mix of 50 μl was prepared by mixing 17 pmol purified mRNAmixture at a ratio of 1:20=MLV_D583N_pD (active RT):del_pD (inactive RT)with 4.3 time of molar excess of long+ and 4.1 time of molar excess ofddC-Long2 in nuclease free water. The mixture was incubated at 70° C.for five min and then cooled to room temperature for 20 min. Beforeadding ligation reaction components, the mixture was moved to a coolingstand for two min.

Five μl of 10× ligation buffer and 5 μl of T4 DNA ligase (5 v/μl)(Fermentas) were added to 40 μl of annealing mixture. Negative ligationreaction was carried out using the same annealing mixture in 1× ligationbuffer without T4 DNA ligase. Prepared ligation reaction mixtures wereincubated at 37° C. for 30 min. To stop ligation, 1 μl 0.5M EDTA (pH8.0) was added to both tubes and reaction mixtures were extracted oncewith equal volume of Roti® Phenol/chloroform (ROTH) and twice with equalvolume of chloroform followed by concentration of reaction products at30° C. for 10 min in vacuum Concentrator 5301 (Eppendorf). Desalting wasperformed using illiustra ProbeQuant G-50 MicroColumns (GE Healthcare).Concentrations of ligation products were determined on agarose gel alongwith RiboRuler™ RNA Ladder, High Range (Fermentas)—mRNA mixture(MLV_D583N_pD:del_pD=1:20) ligated to long+/ddC-Long2oligodeoxynucleotides ˜0.24 μg/μl (sample with T4 DNA ligase) and simplemRNA mixture with long+/ddC-Long2 oligodeoxynucleotides ˜0.06 μg/μl(sample without T4 DNA ligase).

(4) General control of mRNA/dsDNA complex by incorporation of[α-P³³]dATP.

Prepared mRNA/dsDNA complex (substrate) was tested for dTTP (orbiotin-dUTP) and subsequent [α-P³³]dATP incorporation by reversetranscriptase. Reaction mixture: 16 μl of 5× Reaction buffer for reversetranscriptase; 4 μl-40 u/μl RiboLock™ RNase inhibitor (Fermentas); 2 μl[α-P³³]dATP (10 mCi)/ml, SRF-203 (Hartmann Analytic)); 35 μl nucleasefree water; 1 μl-200 u/μl RevertAid™ Minus M-MuLV Reverse transcriptase(Fermentas). Prepared mix was divided to two tubes 2×28 μl and 2 μl of 1mM dTTP (Fermentas) or 2 μl of 1 mM biotin-dUTP (Fermentas) was added.Resultant mixtures were divided into two tubes 2×15μl. To the first tube1.25 μl (˜0.3 μg) of ligation products plus 3.75 μl of nuclease freewater were added. To the second tube 5 μl (˜0.3 μg) of negative ligationreaction products were added. Reaction mixtures were incubated at 37° C.for 30 min. After that, 1 μl of 0.5M EDTA (pH 8.0) was added to alltubes and reaction mixtures were extracted once with equal volume ofRoti® Phenol/chloroform (ROTH) and twice with equal volume of chloroformfollowed by purification on illiustra ProbeQuant G-50 MicroColumns (GEHealthcare). Reactions products were analyzed on agarose gels along withRiboRuler™ RNA Ladder, High Range (Fermentas) FIG. 14A. In all samples(with and without ligase) a discreet band (˜2500 b) of del_pD mRNA wasseen (20 times smaller amount of MLV_D583N_pD mRNA ˜2700 b, which waspresent in mRNA mixture cannot be distinguished).

Subsequently, the agarose gel was dried on filter paper and radiolabeledmRNA/dsDNA complex (at the same position as mRNA) was detected only incase of positive ligation samples (with ligase) and not in case ofnegative ligation samples (without ligase) FIG. 14B.

(5) Selection for DNA dependent DNA polymerase activity usingbiotin-dUTP.

Previously prepared mRNA/dsDNA complex (mRNA mix MLV_D583N_pD (activeRT):del_pD (inactive RT)=1:20) was used for in vitro translationemploying synthetic WakoPURE system (295-59503—Wako). Translationmixture for WakoPURE system (25 μl): 12.5 μl—A solution (Wako); 5 μl—Bsolution (Wako); 0.5 μl-40 u/μl RiboLock RNase inhibitor (Fermentas);0.25 μl-1 M DTT; 1.75 μl nuclease-free water and 5 μl-0.24 μg/μlmRNA/dsDNA substrate (˜1200 ng). In vitro translation was performed for120 min at 37° C.

Translations (˜25 μl) were stopped by adding 155 μl of ice-cold stopbuffer WBK₅₀₀+DTT+Triton (50 mM Tris-acetate pH 7.5 at 25° C.; 50 mMNaCl; 50 mM Mg-acetate; 500 mM KCl; 10 mM DTT; 0.1% (v/v)—Triton X-100(T8787-Sigma)) and centrifuged for five min at +4° C. and 25′000 g. Verycarefully 160 μl of centrifuged translation mixture was transferred onthe top of 840 μl 35% (w/v) sucrose solution in WBK₅₀₀+DTT+Triton X-100(50 mM Tris-acetate pH 7.5 at 25° C.; 50 mM NaCl; 50 mM Mg-acetate; 500mM KCl; 10 mM DTT; 0.1% (v/v)—Triton X-100 (T8787-Sigma); 35%(w/v)—sucrose (84097-Fluka)) to transparent 1 ml ultracentrifugationtubes (343778-Beckman). Ternary complexes (TC) consisting ofmRNA/dsDNA-ribosome-protein (tRNA) were purified by ultracentrifugationat TL-100 Beckman ultracentrifuge in TLA100.2 fixed angle rotor(Beckman) at 100,000 rpm for nine min at +4° C. Initially 750 μl ofsolution was removed from the very top of the centrifugation tube. Then,very carefully to keep small transparent pellet of TC at the bottom ofultracentrifugation tube intact, tube walls were washed with 750 μl ofWBK₅₀₀ (50 mM tris-acetate pH 7.5 at 25° C.; 50 mM NaCl; 50 mMMg-acetate; 500 mM KCl). Finally all solution was removed starting fromthe very top of the centrifugation tube and the pellet was dissolved in30 μl ice-cold stop buffer WBK₅₀₀+DTT+triton (50 mM Tris-acetate pH 7.5at 25° C.; 50 mM NaCl; 50 mM Mg-acetate; 500 mM KCl; 10 mM DTT; 0.1%(v/v)—Triton X-100 (T8787-Sigma)).

As determined using radioactively labeled mRNA after ultracentrifugation5%-30% of input mRNA was located in the ternary complex pellet.Therefore it was predicted to have less than 360 ng (30% from 1200 ngmRNA used in translation reaction) of mRNA in 30 μl buffer (˜12 ng/μl or9*10⁹ molecules/μl of ternary complex).

Modified nucleotide incorporation reaction mix was prepared on ice bymixing: 5 μl-5× reaction buffer for Reverse Transcriptase (Fermentas);1.25 μl-40 u/μl RiboLock RNase inhibitor (Fermentas); 2.5 μl-1 mMbiotin-dUTP (Fermentas); 40.95 μl nuclease-free water and 0.3 μl ofpurified (<2.7*10⁹ molecules) TC. According to the protocol 50 μl ofnucleotide incorporation reaction mix contains <2.7*10⁹ molecules ofternary complex.

Oil-surfactant mixture was prepared by mixing ABIL EM 90 (Goldschmidt)into mineral oil (M5904-Sigma) to a final concentration of 4% (v/v)(Ghadessy and Holliger, 2004; U.S. 2005/064460). Emulsions were preparedat +4° C. in 5 ml cryogenic vials (430492-Corning) by mixing 950 μl ofoil-surfactant mixture with 50 μl of RT mixture. Mixing was performedusing MS-3000 magnetic stirrer with speed control (Biosan) at ˜2100 rpm;Rotilabo®—(3×8 mm) magnetic bar with centre ring (1489.2-Roth); waterphase was added in 10 μl aliquots every 30 sec, continuing mixing fortwo more minutes (total mixing time—4 min). According to opticalmicroscopy data compartments in prepared emulsions vary from 0.5 μm to10 μm in size with average diameter of ˜2 μm. Therefore it was expectedto have ˜1*10¹⁰ water in oil compartments after the emulsification of 50μl reverse transcription reaction mixture, which contains less than2.7*10⁹ molecules of ternary complex (about one mRNA-dsDNA complex andreverse transcriptase molecules per 3-4 compartments).

The prepared emulsion was incubated for 30 min at +37° C.

To recover the reaction mixture from the emulsion, 20 μl 0.1M EDTA wasadded to the emulsion, stirred for ten sec, then 50 μl phenol/chloroformmix was added and stirred for an additional ten sec. After that, theemulsion was transferred to 1.5 ml microcentrifuge tube, 0.5 ml ofwater-saturated ether was added mixed by vortexing and centrifuged forten min at room temperature for 16,000 g. Oil-ether phase was removedleaving concentrated but still intact emulsion at the bottom of thetube. Finally emulsions were broken by extraction with 0.9 mlwater-saturated ether, 0.9 ml water-saturated ethyl-acetate (in order toremove ABIL EM 90 detergent), and twice with 0.9 ml water-saturatedether. The water phase was dried for twelve min under vacuum at roomtemperature followed removal of incorporated nucleotides on illiustraProbeQuant G-50 MicroColumns (GE Healthcare). Two μl aliquot ofresultant mixture was left for direct RT reaction (without streptavidinbeads purification) and the remaining part of the solution was used forbiotinylated mRNA/dsDNA complex immobilization on Dynabeads M-280Streptavidin beads (DYNAL Biotech).

Dynabeads® kilobase BINDER™ Kit (DYNAL Biotech) was used for isolationof biotinylated mRNA-dsDNA complex according to the provided productdescription. Five μl of resuspended Dynabeads was transferred to a 1.5ml microcentrifuge tube, and washed with 20 μl of provided BindingSolution. The tube was placed on a magnet for 1-2 min until beadssettled in the tube and the solution was removed. Dynabeads were gentlyresuspended by pipetting 50 μl of Binding Solution and 1 μl of aqueoustRNA (tRNA from yeast (Roche)) solution (1 μg/μl) was added to minimizenon-specific mRNA binding. Fifty μl Dynabeads in Binding solution wereadded to solution (˜50 μl) containing the biotinylated RNA-DNAfragments. The tube was incubated shaking at +22° C. in termomixer(Eppendorf) for one hour. After the binding of mRNA/dsDNA, supernatantwas removed from the beads, and the beads were washed two times with 50μl of Washing Solution (provided in the kit) shaking (1400 rpm) for fivemin at +22° C. and once with 50 μl of Washing Solution shaking (1400rpm) for twelve min at +22° C. Collected Dynabeads with immobilizedbiotinylated mRNA/dsDNA complex were resuspended in reversetranscription reaction mixture.

Reverse transcription reaction mixture for selected mRNA/dsDNA complexwas prepared on ice: 12 μl-5× reaction buffer for Reverse Transcriptase(Fermentas); 6 μl-10 mM dNTP (Fermentas); 1.5 μl-40 u/μl RiboLock RNaseinhibitor (Fermentas); 0.3 μl-20 μM pD_42 oligonucleotide (SEQ ID No:8); 1.5 u/μl RevertAid™ Minus M-MuLV Reverse transcriptase (200u/μl)(Fermentas); 35.7 μl nuclease-free water. Prepared mix was dividedto three 19 μl aliquots: one was used to resuspend Dynabeads withimmobilized biotinylated mRNA/dsDNA complex, the other aliquot of 19μwas transferred to the tube with sample of elongated mRNA/dsDNA complexleft without streptavidin beads purification and to the rest of 19 μlaliquot was added 1 μl of nuclease free water (negative reactioncontrol—to prove that reaction mixture is not contaminated). Allreaction mixtures were incubated by shaking (1000 rpm) at the +42° C. intermomixer (Eppendorf) for one hour.

Amplification of cDNA was performed by nested PCR. Initial PCR mixturewas prepared on ice: 10 μl-10× Taq buffer with KCl (Fermentas); 10 μl-2mM of each dNTP (Fermentas); 6 μl-25 mM MgCl₂ (Fermentas); 2 μl-1 u/μlLC (recombinant) Taq DNA Polymerase (Fermentas); 1 μl-2.5 u/μl Pfu DNAPolymerase (Fermentas); 0.5 μl-100 μM RD_Nde primer (SEQ ID No: 9); 0.5μl-100 μM pD_55 primer (SEQ ID No: 10); 65 μl water—mixture was dividedinto 5 samples for 19 μl (5×19 μl). To 3×19 μl of PCR master mix wereadded 1 μl of cDNA (1-3 RT samples); to one tube with 19 μl of PCRmaster mix 1 μl water was added—negative PCR control. For positive PCRcontrol 1 μl of pET_his_MLV_pD plasmid (˜1 ng) was added. The cyclingprotocol was: initial denaturation step three min at 94° C., 30 cycles(45 sec at 94° C., 45 sec at 58° C., and three min at 72° C.) and finalelongation for five min at 72° C.

Nested PCR mixture for partial gene amplification (for better resolutionof MLV_D583N_pD:del_pD cDNA ratio in RT samples) was prepared on ice: 20μl-10× Taq buffer with KCl (Fermentas); 20 μl-2 mM of each dNTP(Fermentas); 12 μl-25 mM MgCl₂ (Fermentas); 0.9 μl-5 u/μl Taq DNAPolymerase (Fermentas); 1.0 μl-100 μM M_F primer (SEQ ID No: 11); 1.0μl-100 μM M_2R primer (SEQ ID No: 12); 135.1 μl water-mixture wasdivided 5×38 μl. 2 μl of first PCR (primers set RD_Nde//pD_55) productwere added to prepared nested PCR mixture. Master mix was mixed againand divided into two tubes (2×20 μl) for 30 or 35 PCR cyclesamplification. The cycling protocol was: initial denaturation step 3 minat 94° C., 30 or 35 cycles (45 sec at 94° C., 45 sec at 57° C., and onemin at 72° C.) and final elongation for three min at 72° C. Expectedlength of PCR fragments was 907 bp for MLV_D583N_pD and 736 bp fordel_pD. Amplification was analyzed on 1% agarose gel loading 10 μl ofPCR mix per well (FIG. 15).

Results

1. Double stranded (dsDNA) adaptor was successfully ligated to mRNAusing T4 DNA ligase. Ligation efficiency is about 60% as it wasdetermined by ligation of dsDNA-biotin adapter. mRNA/dsDNA complex couldbe specifically purified on streptavidin beads, providing an opportunityto discriminate between biotin labeled and unlabeled substrates. FreemRNA is a much poorer substrate for DNA dependent DNA polymerasecompared to mRNA/dsDNA. As a consequence 60% ligation efficiency ofdsDNA to mRNA was sufficiently good and such a substrate could besuccessfully used in evolution scheme.2. General selection experiment using mRNA/dsDNA complex (mRNA mixtureMLV_D583N_pD:del_pD=1:20) was performed. In vitro translation wasperformed using WakoPURE protein translation system andcompartmentalized biotin-dUTP incorporation reaction into dsDNA wascarried out to demonstrate the enrichment of genes encoding active(MLV_D583N_pD) reverse transcriptase over genes encoding inactivatedenzyme (del_pD). According to the selection scheme (FIG. 12)incorporation reaction of biotin-dUTP should occur only in aqueouscompartments containing active (MLV_D583N_pD) reverse transcriptaseresulting in biotinylation of mRNA/dsDNA complex. DNA dependent DNApolymerase was selected by binding the biotinylated complex tostreptavidin immobilized on magnetic beads, and then the selected genewas amplified by RT-PCR. Genes encoding active enzyme (in our caseRT-PCR fragment for MLV_D583N_pD reverse transcriptase) were enrichedover genes encoding inactive enzyme (FIG. 15). Initial ratio of genesMLV_D583N_pD:del_pD was 1:20 and final ratio (after enrichment) was˜1:1. Respectively an enrichment factor in this particular experimentwas ˜20 folds. Enrichment factors calculated from different experimentsvaried in range from 5 to 200. It was confirmed that DNA dependent DNApolymerase could be selected for modified nucleotide incorporationapplying the Compartmentalized Ribosome Display (CRD) method. Selectionof conventional DNA dependent DNA polymerases could be performedstraightaway. Biotin-dUTP could be exchanged to different nucleotideanalogues of interest including nucleotide analogues having 3′modifications. After the incorporation of such nucleotide analogues intoDNA strand the 3′ end is blocked, cannot be extended and elongationreaction will be terminated. This approach was used in sequencing bysynthesis (SBS) scheme and DNA polymerase suitable for SBS can be easilyevolved using compartmentalized ribosome display (CRD) technique.

APPENDIX 1 (SEQ ID NO: 24) NcoIccatgggcatgaccctaaatatagaagatgagcatcggctacatgagacctcaaaagagc   60cagatgtttctctagggtccacatggctgtctgattttcctcaggcctgggcggaaaccg  120ggggcatgggactggcagttcgccaagctcctctgatcatacctctgaaagcaacctcta  180cccccgtgtccataaaacaataccccatgtcacaagaagccagactggggatcaagcccc  240                            g                         gacatacagagactgttggaccagggaatactggtaccctgccagtccccctggaacacgc  300                                        accctgctacccgttaagaaaccagggactaatgattataggcctgtccaggatctgagag  360                     g                    gaagtcaacaagcgggtggaagacatccaccccaccgtgcccaacccttacaacctcttga  420              cgcgggctcccaccgtcccaccagtggtacactgtgcttgatttaaaggatgcctttttct  480         c gcctgagactccaccccaccagtcagcctctcttcgcctttgagtggagagatccagaga 540 tgggaatctcaggacaattgacctggaccagactcccacagggtttcaaaaacagtccca  600                     c                            t   tccctgtttgatgaggcactgcacagagacctagcagacttccggatccagcacccagact  660            gtgatcctgctacagtacgtggatgacttactgctggccgccacttctgagctagactgcc  720aacaaggtactcgggccctgttacaaaccctagggaacctcgggtatcgggcctcggcca  780          g                         cagaaagcccaaatttgccagaaacaggtcaagtatctggggtatcttctaaaagagggtc  840agagatggctgactgaggccagaaaagagactgtgatggggcagcctactccgaagaccc  900                                                actcgacaactaagggagttcctagggacggcaggcttctgtcgcctctggatccctgggt  960ttgcagaaatggcagcccccttgtaccctctcaccaaaacggggactctgtttaattggg 1020gcccagaccaacaaaaggcctatcaagaaatcaagcaagctcttctaactgccccagccc 1080                                 ctggggttgccagatttgactaagccctttgaactctttgtcgacgagaagcagggctacg 1140ccaaaggtgtcctaacgcaaaaactgggaccttggcgtcggccggtggcctacctgtcca 1200aaaagctagacccagtagcagctgggtggcccccttgcctacggatggtagcagccattg 1260ccgtactgacaaaggatgcaggcaagctaaccatgggacagccactagtcattctggccc 1320  -------                         ---------------cccatgcagtagaggcactagtcaaacaaccccccgaccgctggctttccaacgcccgga 1380tgactcactatcaggccttgcttttggacacggaccgggtccagttcggaccggtggtag 1440ccctgaacccggctacgctgctcccactgcctgaggaagggctgcaacacaactgccttg 1500             gatatcctggccgaagcccacggaacccgacccgacctaacggaccagccgctcccagacg 1560                               c        gccgaccacacctggtacacggatggaagcagtctcttacaagagggacagcgtaaggcgg 1620gagctgcggtgaccaccgagaccgaggtaatctgggctaaagccctgccagccgggacat 1680ccgctcagcgggctgaactgatagcactcacccaggccctaaagatggcagaaggtaaga 1740                                                   gagctaaatgtttatactgatagccgttatgcttttgctactgcccatatccatggagaaa 1800tatacagaaggcgtgggttgctcacatcagaaggcaaagagatcaaaaataaagacgaga 1860tcttggccctactaaaagccctctttctgcccaaaagacttagcataatccattgtccag 1920            a                       ggacatcaaaagggacacagcgccgaggctagaggcaaccggatggctgaccaagcggccc 1980gaaaggcagccatcacagagactccagacacctctaccctcctcatagaaaattcatcac 2040ccaattcccgcttaattaatgaattc                      EcoRI

APPENDIX 2 L5_9 12 mutations, 83D->N, 135L->P, 166P->S, 214H->R,222Y->C, 293T->A, 344Y->H, 407P->L, 415A->V, 436A->T, 444V->A, 447P->L18 11 mutations, 83D->N, 139L->P, 200D->N, 330T->P, 479N->D, 577K->Q,583D->G, 618L->V, 678N->I, 679S->P, 680R->A L5_79 10 mutations, 74I->T,104P->R, 325Y->H, 333L->Q, 430Q->R, 597I->T, 616E->K, 649N->S, 658K->R,673E->G L5_82 10 mutations, 26L->P, 130P->S, 137S->G, 343A->T, 356A->G,524D->G, 539A->T, 603L->W, 653D->N, 676S->P L5_117 10 mutations, 14S->P,95N->S, 139L->P, 190Q->R, 333L->Q, 339D->G, 380V->A, 383Q->P, 524D->G,532G->R 21  9 mutations, 131N->S, 179I->T, 204H->R, 323P->L, 353T->A,583D->A, 678N->I, 679S->P, 680R->A L5_3  9 mutations, 125I->V, 138G->R,143H->R, 380V->A, 552L->P, 603L->W, 622K->R, 658K->Q, 668S->P L5_20  9mutations, 12E->A, 87L->P, 95N->S, 200D->A, 221Q->R, 242A->T, 331G->E,428M->L, 603L->W L5_75  9 mutations, 11H->Y, 325Y->H, 333L->Q, 430Q->R,597I->T, 616E->K, 649N->S, 658K->R, 673E->G L5_76  9 mutations, 66M->L,105G->E, 200D->N, 289M->V, 314I->T, 436A->T, 491L->P, 573A->T, 583D->N 8 8 mutations, 89P->S, 139L->P, 287T->A, 330T->P, 514L->F, 607E->K,635C->S, 663E->D L5_13  8 mutations, 130P->S, 159R->K, 200D->A, 221Q->R,330T->P, 449D->G, 524D->G, 583D->E L5_41  8 mutations, 60S->A, 139L->P,168F->S, 199F->Y, 346E->D, 450R->H, 524D->G, 583D->G L5_68  8 mutations,39M->V, 49I->V, 91Q->R, 204H->R, 287T->A, 454N->K, 625F->L, 653D->HL5_72  8 mutations, 139L->P, 200D->N, 330T->P, 393A->T, 572M->L,594H->Q, 671L->P, 672I->T L5_114  8 mutations, 17P->S, 108D->E, 249N->D,307A->V, 344Y->H, 524D->G, 653D->G, 673E->K L5_115  8 mutations, 8H->P,139L->P, 197T->A, 200D->G, 358G->W, 524D->G, 623A->V, 653D->A 17  7mutations, 49I->V, 65P->S, 200D->N, 409C->R, 470V->A, 502A->V, 583D->NL5_15  7 mutations, 17P->S, 108D->E, 249N->D, 307A->V, 344Y->H, 524D->G,653D->G L5_21, L5_111  7 mutations, 65P->S, 233E->K, 407P->S, 478L->P,603L->W, 638H->R, 653D->N L5_40  7 mutations, 24T->A, 41L->R, 127P->S,151L->F, 330T->P, 503H->R, 653D->G L5_47  7 mutations, 107N->S, 126H->R,128T->A, 179I->V, 200D->N, 642H->Y, 653D->N L5_71  7 mutations, 97P->S,184T->A, 250L->P, 289M->L, 497D->G, 551A->T, 562E->K L5_78  7 mutations,11H->R, 148V->M, 330T->P, 459H->R, 502A->V, 653D->G, 667T->A L5_81  7mutations, 166P->S, 199F->L, 446Q->R, 468D->A, 501E->K, 530Q->H, 558A->VL5_118  7 mutations, 221Q->R, 283A->T, 287T->A, 369F->I, 376Y->C,434I->T, 603L->W 13  6 mutations, 51P->S, 136L->W, 207L->P, 428M->L,560R->W, 603L->W L5_30  6 mutations, 200D->A, 330T->P, 449D->G, 479N->D,583D->N, 671L->P L5_32  6 mutations, 69E->G, 135L->P, 139L->P, 431P->Q,583D->G, 679S->F L5_43, L5_112  6 mutations, 200D->N, 237Q->R, 330T->P,524D->G, 625F->S, 653D->N L5_53  6 mutations, 130P->S, 139L->P, 417A->V,524D->G, 583D->N, 634H->Y L5_56  6 mutations, 8H->P, 211R->W, 292P->L,486L->P, 524D->A, 594H->R L5_57  6 mutations, 49I->V, 173R->K, 302E->K,392V->A, 603L->W, 669T->S L5_60  6 mutations, 112V->A, 200D->N, 280L->P,322A->T, 379G->S, 653D->N L5_62  6 mutations, 118V->A, 204H->R, 282E->G,346E->D, 524D->A, 528L->I L5_84  6 mutations, 126H->R, 139L->P, 417A->V,491L->P, 524D->G, 653D->A L5_95  6 mutations, 124D->G, 187R->G, 263Q->R,494N->K, 583D->G, 618L->V L5_107  6 mutations, 139L->P, 233E->K,295K->E, 633I->T, 642H->R, 643S 16  5 mutations, 241R->Q, 259A->T,311R->H, 544T->I, 656A->T 20  5 mutations, 200D->N, 603L->W, 678N->I,679S->P, 680R->A 23  5 mutations, 221Q->R, 332T->I, 524D->A, 644A->V,661I->V L5_2  5 mutations, 5E->K, 43V->I, 184T->A, 391P->L, 543E->KL5_14  5 mutations, 37G->W, 197T->A, 200D->G, 433V->A, 603L->W L5_18  5mutations, 457M->T, 462A->T, 504G->R, 559Q->R, 655A->V L5_23  5mutations, 30P->L, 223V->M, 388W->R, 390R->W, 435L->P L5_24  5mutations, 139L->P, 221Q->R, 603L->W, 649N->S, 658K->R L5_28  5mutations, 306T->A, 309F->S, 524D->A, 594H->R, 625F->S L5_37  5mutations, 126H->S, 149L->F, 200D->N, 454N->K, 583D->N L5_49, L5_63  5mutations, 50I->V, 194N->S, 204H->R, 287T->A, 524D->A L5_55  5mutations, 5E->K, 200D->N, 240T->A, 653D->H, 671L->P L5_69  5 mutations,14S->T, 49I->T, 538A->T, 603L->W, 653D->N L5_88  5 mutations, 70A->V,139L->P, 479N->D, 524D->G, 625F->L L5_101  5 mutations, 65P->S, 204H->R,283A->D, 391P->S, 583D->N L5_104  5 mutations, 132P->S, 164S->G,388W->R, 524D->G, 533Q->K 3  4 mutations, 221Q->R, 428M->L, 602G->R,603L->W 5  4 mutations, 139L->P, 283A->D, 358G->V, 653D->N 7  4mutations, 204H->R, 433V->A, 524D->G, 572M->I 11  4 mutations, 29F->L,148V->M, 390R->W, 653D->A 12  4 mutations, 23S->P, 88V->A, 237Q->R,623A->V 30  4 mutations, 200D->N, 306T->M, 524D->N, 583D->G L5_1  4mutations, 249N->D, 409C->R, 470V->A, 502A->V L5_6  4 mutations,287T->A, 524D->A, 594H->R, 680R->P L5_8  4 mutations, 475V->G, 524D->G,679S->P, 680R->A L5_11  4 mutations, 15K->T, 200D->N, 576K->R, 607E->AL5_16  4 mutations, 91Q->L, 583D->G, 600R->K, 603L->M L5_29  4mutations, 174D->G, 312L->P, 502A->V, 524D->G L5_39  4 mutations,12E->V, 86I->V, 200D->N, 646A->V L5_42  4 mutations, 192F->L, 333L->P,556T->A, 603L->W L5_44  4 mutations, 92S->P, 430Q->R, 479N->D, 583D->NL5_46  4 mutations, 200D->N, 330T->P, 583D->N, 644A->T L5_52  4mutations, 200D->N, 330T->P, 374Q->R, 583D->N L5_61  4 mutations,64Y->C, 351L->V, 449D->A, 530Q->H L5_64, L5_93  4 mutations, 200D->N,216D->G, 524D->A, 545E->G L5_65  4 mutations, 200D->N, 238Q->H, 570L->I,603L->W L5_85, L5_96  4 mutations, 200D->N, 330T->P, 583D->A, 638H->RL5_90  4 mutations, 200D->N, 298R->G, 330T->P, 374Q->R L5_103  4mutations, 96T->M, 200D->N, 559Q->P, 607E->G L5_106  4 mutations,524D->G, 583D->N, 635C->R, 670L->F L5_120  4 mutations, 252Y->H,308G->S, 441E->G, 603L->W 1  3 mutations, 49I->V, 524D->A, 594H->R 28  3mutations, 8H->R, 632I->T, 644A->V L5_4  3 mutations, 208A->V, 225D->G,680R->A L5_25  3 mutations, 39M->L, 302E->K, 628K->E L5_35  3 mutations,200D->N, 330T->P, 479N->D L5_51  3 mutations, 110R->G, 431P->Q, 653D->NL5_58  3 mutations, 66M->L, 90C->Y, 653D->V L5_66  3 mutations, 93P->L,457M->R, 603L->W L5_73  3 mutations, 67S->P, 139L->P, 307A->V L5_80  3mutations, 326P->S, 583D->G, 676S->P L5_92  3 mutations, 126H->S,200D->N, 653D->G L5_94  3 mutations, 494N->D, 524D->G, 607E->K L5_97  3mutations, 484L->P, 498I->V, 653D->A 10  2 mutations, 481A->T, 524D->GL5_59  2 mutations, 450R->H, 503H->R L5_99  2 mutations, 36T->I, 524D->GL5_116  2 mutations, 653D->H, 662T->A L5_48  1 mutations, 77H->RVVT_MLV, 0 mutations L5_87

APPENDIX 3 SEQ ID No: 1 LOCUSpET-his-MLV-pD 7873 bp DNA circular 5 Jun. 2007 SOURCE ORGANISM COMMENTThis file is created by Vector NTI http://www.invitrogen.com/ COMMENTORIGDB|GenBank COMMENT VNTDATE|448383004| COMMENT VNTDBDATE|448383004|COMMENT LSOWNER| COMMENT VNTNAME|pET-his-MLV-PD| COMMENTVNTAUTHORNAME|Remigijus Skirgaila| COMMENT VNTAUTHORTEL|+370-5-2394224|COMMENT VNTAUTHOREML|skirgaila@fermentas.lt| COMMENTVNTAUTHORWWW|www.fermentas.com| FEATURES Location/Qualifiers CDS3534..4391 /vntifkey = “4” /label = Ap /note =“ORF: Frame #2 Start: atg Stop: taa” CDS complement(6586..7674)/vntifkey = “4” /label = lacl /note =“ORF: Frame #3 Start: gtg Stop: tga” terminator 2864..2910 /vntifkey =“43” /label = T7\terminator rep_origin 2947..3402 /vntifkey = “33”/label = f1\origin rep_origin complement(2936..2936) /vntifkey = “33”/label = ori promoter 174..190 /vntifkey = “30” /label = PT7misc_feature 2394..2669 /vntifkey = “21” /label = pD misc_feature2364..2393 /vntifkey = “21” /label = gs\linker misc_feature 2670..2759/vntifkey = “21” /label = gs\linker misc_feature 306..2363 /vntifkey =“21” /label = MLV\H+ misc_feature 258..305 /vntifkey = “21” /label = hisBASE COUNT 1873 a 2155 c 2084 g 1761 t ORIGIN    1cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc   61ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc  121ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctatacga aattaatacg  181actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga  241aagaggagaa attacatatg agaggatcgc atcaccatca ccatcacgga tctggttcca  301tgggcatgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag  361atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg  421gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc  481ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca  541tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc  601tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag  661tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg  721ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc  781tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg  841gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc  901tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga  961tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 1021aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc tcggccaaga 1081aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa gagggtcaga 1141gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg aagacccctc 1201gacaactaag ggagttccta gggacggcag gcttctgtcg cctctggatc cctgggtttg 1261cagaaatggc agcccccttg taccctctca ccaaaacggg gactctgttt aattggggcc 1321cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc ccagccctgg 1381ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag ggctacgcca 1441aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac ctgtccaaaa 1501agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca gccattgccg 1561tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt ctggcccccc 1621atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac gcccggatga 1681ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg gtggtagccc 1741tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac tgccttgata 1801tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc ccagacgccg 1861accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt aaggcgggag 1921ctgcggtgac caccgagacc gaggtaatct gggctaaagc cctgccagcc gggacatccg 1981ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa ggtaagaagc 2041taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat ggagaaatat 2101acagaaggcg tgggttgctc acatcagaag gcaaagagat caaaaataaa gacgagatct 2161tggccctact aaaagccctc tttctgccca aaagacttag cataatccat tgtccaggac 2221atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa gcggcccgaa 2281aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat tcatcaccca 2341attcccgctt aattaatgaa ttcggatccg gtggcggttc cggcggtgga tctatgggta 2401ccgcaaccgc gcccggcgga ttgagtgcga aagcgcctgc aatgaccccg ctgatgctgg 2461acacctccag ccgtaagctg gttgcgtggg atggcaccac cgacggtgct gccgttggca 2521ttcttgcggt tgctgctgac cagaccagca ccacgctgac gttctacaag tccggcacgt 2581tccgttatga ggatgtgctc tggccggagg ctgccagcga cgagacgaaa aaacggaccg 2641cgtttgccgg aacggcaatc agcatcgttg gatctggtgg cggttccggc ggtggatctg 2701gtggcggttc cggcggtgga tctggtggcg gttccggcgg tggatcgtgt cttctttaag 2761cttgcggccg cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc 2821cgaaaggaag ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg 2881gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg 2941aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg 3001tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc 3061tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc 3121gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta 3181gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta 3241atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg 3301atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa 3361aatttaacgc gaattttaac aaaatattaa cgtttacaat ttcaggtggc acttttcggg 3421gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 3481tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 3541ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 3601ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 3661gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 3721gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg 3781acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 3841actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 3901ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 3961cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 4021gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag 4081caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 4141aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 4201ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 4261tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 4321ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 4381ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 4441ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 4501tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 4561cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 4621taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 4681gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 4741acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 4801ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 4861ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 4921cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 4981aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 5041gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 5101gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 5161gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 5221ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 5281ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 5341tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata tatggtgcac 5401tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta 5461cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg 5521gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg 5581tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca 5641gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt 5701ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt 5761tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata 5821ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta 5881ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc 5941actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag 6001cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc 6061agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt 6121ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta 6181aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt 6241ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca 6301gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc 6361atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc 6421tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc 6481cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc 6541ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag 6601tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 6661ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt 6721gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag 6781caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt 6841atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc 6901gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc 6961ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg 7021ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag 7081acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc 7141gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat 7201gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac 7261agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc 7321gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac 7381caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg 7441cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag 7501ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc 7561ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga 7621gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa 7681ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt 7741gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta 7801ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca 7861acagtccccc ggc SEQ ID No: 2 LOCUSpET_his_del_pD 7702 bp DNA circular 6 Jun. 2007 SOURCE ORGANISM COMMENTThis file is created by Vector NTI http://www.invitrogen.com/ COMMENTVNTDATE|448455242| COMMENT VNTDBDATE|448455855| COMMENT LSOWNER| COMMENTVNTNAME|pET_his_del_PD| COMMENT VNTAUTHORNAME|Remigijus Skirgaila|COMMENT VNTAUTHORTEL|+370-5-2394224| COMMENTVNTAUTHOREML|skirgaila@fermentas.lt| COMMENTVNTAUTHORWWW|www.fermentas.com| FEATURES Location/Qualifiersmisc_feature 2499..2588 /vntifkey = “21” /label = gs\linker misc_feature2193..2222 /vntifkey = “21” /label = gs\linker misc_feature 2223..2498/vntifkey = “21” /label = pD promoter 174..190 /vntifkey = “30” /label =PT7 rep_origin complement(2765..2765) /vntifkey = “33” /label = orirep_origin 2776..3231 /vntifkey = “33” /label = f1\origin terminator2693..2739 /vntifkey = “43” /label = T7\terminator CDScomplement(6415..7503) /vntifkey = “4” /label = lacl /note =“ORF: Frame #3 Start: gtg Stop: tga” CDS 3363..4220 /vntifkey = “4”/label = Ap /note = “ORF: Frame #2 Start: atg Stop: taa” misc_feature258..305 /vntifkey = “21” /label = his misc_feature 306..2192/vntifkey = “21” /label = del BASE COUNT 1823 a 2115 c 2033 g 1731 tORIGIN    1cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc   61ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc  121ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctatacga aattaatacg  181actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga  241aagaggagaa attacatatg agaggatcgc atcaccatca ccatcacgga tctggttcca  301tgggcatgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag  361atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg  421gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc  481ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca  541tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc  601tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag  661tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg  721ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc  781tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg  841gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc  901tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga  961tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 1021aaggtactcg ggccctgtta caaaccctag ggacggcagg cttctgtcgc ctctggatcc 1081ctgggtttgc agaaatggca gcccccttgt accctctcac caaaacgggg actctgttta 1141attggggccc agaccaacaa aaggcctatc aagaaatcaa gcaagctctt ctaactgccc 1201cagccctggg gttgccagat ttgactaagc cctttgaact ctttgtcgac gagaagcagg 1261gctacgccaa aggtgtccta acgcaaaaac tgggaccttg gcgtcggccg gtggcctacc 1321tgtccaaaaa gctagaccca gtagcagctg ggtggccccc ttgcctacgg atggtagcag 1381ccattgccgt actgacaaag gatgcaggca agctaaccat gggacagcca ctagtcattc 1441tggcccccca tgcagtagag gcactagtca aacaaccccc cgaccgctgg ctttccaacg 1501cccggatgac tcactatcag gccttgcttt tggacacgga ccgggtccag ttcggaccgg 1561tggtagccct gaacccggct acgctgctcc cactgcctga ggaagggctg caacacaact 1621gccttgatat cctggccgaa gcccacggaa cccgacccga cctaacggac cagccgctcc 1681cagacgccga ccacacctgg tacacggatg gaagcagtct cttacaagag ggacagcgta 1741aggcgggagc tgcggtgacc accgagaccg aggtaatctg ggctaaagcc ctgccagccg 1801ggacatccgc tcagcgggct gaactgatag cactcaccca ggccctaaag atggcagaag 1861gtaagaagct aaatgtttat actaatagcc gttatgcttt tgctactgcc catatccatg 1921gagaaatata cagaaggcgt gggttgctca catcagaagg caaagagatc aaaaataaag 1981acgagatctt ggccctacta aaagccctct ttctgcccaa aagacttagc ataatccatt 2041gtccaggaca tcaaaaggga cacagcgccg aggctagagg caaccggatg gctgaccaag 2101cggcccgaaa ggcagccatc acagagactc cagacacctc taccctcctc atagaaaatt 2161catcacccaa ttcccgctta attaatgaat tcggatccgg tggcggttcc ggcggtggat 2221ctatgggtac cgcaaccgcg cccggcggat tgagtgcgaa agcgcctgca atgaccccgc 2281tgatgctgga cacctccagc cgtaagctgg ttgcgtggga tggcaccacc gacggtgctg 2341ccgttggcat tcttgcggtt gctgctgacc agaccagcac cacgctgacg ttctacaagt 2401ccggcacgtt ccgttatgag gatgtgctct ggccggaggc tgccagcgac gagacgaaaa 2461aacggaccgc gtttgccgga acggcaatca gcatcgttgg atctggtggc ggttccggcg 2521gtggatctgg tggcggttcc ggcggtggat ctggtggcgg ttccggcggt ggatcgtgtc 2581ttctttaagc ttgcggccgc actcgagcac caccaccacc accactgaga tccggctgct 2641aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa 2701ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc 2761ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta 2821cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc 2881cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt 2941tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg 3001gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca 3061cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct 3121attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga 3181tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt tcaggtggca 3241cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata 3301tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga 3361gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc 3421ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg 3481cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc 3541ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat 3601cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact 3661tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat 3721tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga 3781tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc 3841ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga 3901tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag 3961cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc 4021gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt 4081ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct 4141acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg 4201cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg 4261atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca 4321tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 4381tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 4441aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 4501aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt 4561taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 4621taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat 4681agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct 4741tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca 4801cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 4861agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 4921gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga 4981aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 5041tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 5101ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg 5161aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat 5221atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg 5281ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg 5341ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg 5401agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa 5461agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc 5521tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg 5581gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg 5641gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat 5701gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag 5761agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag 5821ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc 5881cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc 5941gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc 6001taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg 6061cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg 6121gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac 6181aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct 6241gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata 6301gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt 6361cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc 6421gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 6481agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa 6541cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt 6601ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct 6661gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc 6721ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg 6781aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc 6841gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc 6901cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg 6961acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat 7021actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc 7081agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac 7141gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac 7201catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat 7261ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt 7321gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc 7381cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt 7441ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac 7501caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca 7561ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc 7621ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga 7681tggcgcccaa cagtcccccg gc SEQ ID No: 3 oligonucleotide (22 b) pro-pIVEX5′-GCGAGCCCGATCTTCCCCATCG-3′ SEQ ID No: 4 oligonucleotide (21 b) pD-ter5′-AAGAAGACACGATCCACCGCC-3′ SEQ ID No: 5 oligonucleotide (38 b) assrA5′-TTAAGCTGCTAAAGCGTAGTTTTCGTCGTTTGCGACTA-3′ SEQ ID No: 6protein (833 aa) MLV_pDmrgshhhhhhgsgsmgmtlniedehrlhetskepdvslgstwlsdfpqawaetggmglavrqapliiplkatstpvsikqypmsqearlgikphiqrlldqgilvpcqspwntpllpvkkpgtndyrpvqdlrevnkrvedihptvpnpynllsglppshqwytvldlkdaffclrlhptsqplfafewrdpemgisgqltwtrlpqgfkrisptlfdealhrdladfriqhpdlillqyvddlllaatseldcqqgtrallqtlgnlgyrasakkaqicqkqvkylgyllkegqrwltearketvmgqptpktprqlreflgtagfcrlwipgfaemaaplypltktgtlfnwgpdqqkayqeikqalltapalglpdltkpfelfvdekqgyakgvltqklgpwrrpvaylskkldpvaagwppclrmvaaiavltkdagkltmgqplvilaphavealvkqppdrwlsnarmthyqallldtdrvqfgpwalnpatllplpeeglqhncldilaeahgtrpdltdqplpdadhtwytdgssllqegqrkagaavtteteviwakalpagtsaqraelialtqalkmaegkklnvytdsryafatahihgeiyrrrglltsegkeiknkdeilallkalflpkrlsiihcpghqkghsaeargnrmadqaarkaaitetpdtstlliensspnsrlinefgsgggsgggsmgtatapgglsakapamtplmldtssrklvawdgttdgaavgilavaadqtsttltfyksgtfryedvlwpeaasdetkkrtafagtaisivgsgggsgggsgggsgggsgggsgggscll SEQ ID No: 7protein (766 aa) del_pDmrgshhhhhhgsgsmgmtlniedehrlhetskepdvslgstwlsdfpqawaetggmglavrqapliiplkatstpvsikqypmsqearlgikphiqrlldqgilvpcqspwntpllpvkkpgtndyrpvqdlrevnkrvedihptvpnpynllsglppshqwytvldlkdaffclrlhptsqplfafewrdpemgisgqltwtrlpqgfkrisptlfdealhrdladfriqhpdlillqyvddlllaatseldcqqgtrallqtlgtagfcrlwipgfaemaaplypltktgtlfnwgpdqqkayqeikqalltapalglpdltkpfelfvdekqgyakgvltqklgpwrrpvaylskkldpvaagwppclrmvaaiavltkdagkltmgqplvilaphavealvkqppdrwlsnarmthyqallldtdrvqfgpwalnpatllplpeeglqhncldilaeahgtrpdltdqplpdadhtwytdgssllqegqrkagaavtteteviwakalpagtsaqraelialtqalkmaegkklnvytdsryafatahihgeiyrrrglltsegkeiknkdeilallkalflpkrlsiihcpghqkghsaeargnrmadqaarkaaitetpdtstlliensspnsrlinefgsgggsgggsmgtatapgglsakapamtplmldtssrklvawdgttdgaavgilavaadqtsttltfyksgtfryedvlwpeaasdetkkrtafagtaisivgsgggsgggsgggsgggsgggsgggscllSEQ ID No: 8 oligonucleotide (15 b) pD_42 5′-TTACGGCTGGAGGTG-3′SEQ ID No: 9 oligonucleotide (28 b) RD_Nde5′-CTTTAAGAAAGAGGAGAAATTACATATG-3′ SEQ ID No: 10 oligonucleotide (14 b)pD_55 5′-GCCGGGCGCGGTTG-3′ SEQ ID No: 11 oligonucleotide (23 b) M_F5′-GATCAAGCCCCACATACAGAGAC-3′ SEQ ID No: 12 oligonucleotide (19 b) M_2R5′-GCCCTGCTTCTCGTCGACA-3′ SEQ ID No: 13 oligonucleotide (40 b) M_Esp5′-ATCGTCTCCCATGGGCATGACCCTAAATATAGAAGATGAG-3′ SEQ ID No: 14oligonucleotide (32 b) M_Eri 5′-AATGAATTCATTAATTAAGCGGGAATTGGGTG-3′SEQ ID No: 15 oligonucleotide (18 b) M_1R 5′-CAGGGCCCGAGTACCTTG-3′SEQ ID No: 16 oligonucleotide (17 b) M_3F 5′-CCAGTTCGGACCGGTGG-3′SEQ ID No: 17 oligonucleotide (19 b) pD_ter- 5′-AAGAAGACACGATCCACCG-3′SEQ ID No: 18 oligonucleotide (36 b) M_Hind3+5′-CGGATCAAGCTTAATTAATTAAGCGGGAATTGGGTG-3′ SEQ ID No: 19 LOCUSpET_his_MLV 7474 bp DNA circular 4 Jun. 2007 SOURCE ORGANISM COMMENTThis file is created by Vector NTI http://www.invitrogen.com/ COMMENTORIGDB|GenBank COMMENT VNTDATE|446486240| COMMENT VNTDBDATE|448293778|COMMENT LSOWNER| COMMENT VNTNAME|pET_his_MLV| COMMENTVNTAUTHORNAME|Remigijus Skirgaila| COMMENT VNTAUTHORTEL|+370-5-2394224|COMMENT VNTAUTHOREML|skirgaila@fermentas.lt| COMMENTVNTAUTHORWWW|www.fermentas.com| FEATURES Location/Qualifiers CDS3135..3992 /vntifkey = “4” /label = Ap /note =“ORF: Frame #2 Start: atg Stop: taa” CDS complement(6187..7275)/vntifkey = “4” /label = lacl /note =“ORF: Frame #3 Start: gtg Stop: tga” terminator 2465..2511 /vntifkey =“43” /label = T7\terminator rep_origin 2548..3003 /vntifkey = “33”/label = f1\origin rep_origin complement(2537..2537) /vntifkey = “33”/label = ori promoter 174..190 /vntifkey = “30” /label = PT7 CDS306..2360 /vntifkey = “4” /label = MLV CDS 258..305 /vntifkey = “4”/label = his BASE COUNT 1810 a 2046 c 1942 g 1676 t ORIGIN    1cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc   61ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc  121ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctatacga aattaatacg  181actcactata gggagaccac aacggtttcc ctctagaaat aattttgttt aactttaaga  241aagaggagaa attacatatg agaggatcgc atcaccatca ccatcacgga tctggttcca  301tgggcatgac cctaaatata gaagatgagc atcggctaca tgagacctca aaagagccag  361atgtttctct agggtccaca tggctgtctg attttcctca ggcctgggcg gaaaccgggg  421gcatgggact ggcagttcgc caagctcctc tgatcatacc tctgaaagca acctctaccc  481ccgtgtccat aaaacaatac cccatgtcac aagaagccag actggggatc aagccccaca  541tacagagact gttggaccag ggaatactgg taccctgcca gtccccctgg aacacgcccc  601tgctacccgt taagaaacca gggactaatg attataggcc tgtccaggat ctgagagaag  661tcaacaagcg ggtggaagac atccacccca ccgtgcccaa cccttacaac ctcttgagcg  721ggctcccacc gtcccaccag tggtacactg tgcttgattt aaaggatgcc tttttctgcc  781tgagactcca ccccaccagt cagcctctct tcgcctttga gtggagagat ccagagatgg  841gaatctcagg acaattgacc tggaccagac tcccacaggg tttcaaaaac agtcccaccc  901tgtttgatga ggcactgcac agagacctag cagacttccg gatccagcac ccagacttga  961tcctgctaca gtacgtggat gacttactgc tggccgccac ttctgagcta gactgccaac 1021aaggtactcg ggccctgtta caaaccctag ggaacctcgg gtatcgggcc tcggccaaga 1081aagcccaaat ttgccagaaa caggtcaagt atctggggta tcttctaaaa gagggtcaga 1141gatggctgac tgaggccaga aaagagactg tgatggggca gcctactccg aagacccctc 1201gacaactaag ggagttccta gggacggcag gcttctgtcg cctctggatc cctgggtttg 1261cagaaatggc agcccccttg taccctctca ccaaaacggg gactctgttt aattggggcc 1321cagaccaaca aaaggcctat caagaaatca agcaagctct tctaactgcc ccagccctgg 1381ggttgccaga tttgactaag ccctttgaac tctttgtcga cgagaagcag ggctacgcca 1441aaggtgtcct aacgcaaaaa ctgggacctt ggcgtcggcc ggtggcctac ctgtccaaaa 1501agctagaccc agtagcagct gggtggcccc cttgcctacg gatggtagca gccattgccg 1561tactgacaaa ggatgcaggc aagctaacca tgggacagcc actagtcatt ctggcccccc 1621atgcagtaga ggcactagtc aaacaacccc ccgaccgctg gctttccaac gcccggatga 1681ctcactatca ggccttgctt ttggacacgg accgggtcca gttcggaccg gtggtagccc 1741tgaacccggc tacgctgctc ccactgcctg aggaagggct gcaacacaac tgccttgata 1801tcctggccga agcccacgga acccgacccg acctaacgga ccagccgctc ccagacgccg 1861accacacctg gtacacggat ggaagcagtc tcttacaaga gggacagcgt aaggcgggag 1921ctgcggtgac caccgagacc gaggtaatct gggctaaagc cctgccagcc gggacatccg 1981ctcagcgggc tgaactgata gcactcaccc aggccctaaa gatggcagaa ggtaagaagc 2041taaatgttta tactgatagc cgttatgctt ttgctactgc ccatatccat ggagaaatat 2101acagaaggcg tgggttgctc acatcagaag gcaaagagat caaaaataaa gacgagatct 2161tggccctact aaaagccctc tttctgccca aaagacttag cataatccat tgtccaggac 2221atcaaaaggg acacagcgcc gaggctagag gcaaccggat ggctgaccaa gcggcccgaa 2281aggcagccat cacagagact ccagacacct ctaccctcct catagaaaat tcatcaccca 2341attcccgctt aattaattaa gcttgcggcc gcactcgagc accaccacca ccaccactga 2401gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa 2461taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga 2521ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg 2581gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 2641tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 2701gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 2761attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 2821cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 2881ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 2941aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 3001tttcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa 3061tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt 3121gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg 3181cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag 3241atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg 3301agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg 3361gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt 3421ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga 3481cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac 3541ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc 3601atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc 3661gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac 3721tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag 3781gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg 3841gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta 3901tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg 3961ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata 4021tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt 4081ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc 4141ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct 4201tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa 4261ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag 4321tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc 4381tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg 4441actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca 4501cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat 4561gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg 4621tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc 4681ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc 4741ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc 4801cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg 4861cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga 4921gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt 4981cacaccgcat atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 5041gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca 5101cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 5161accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg 5221cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca 5281tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg 5341gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt 5401ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact 5461gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc ggtatggatg 5521cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa tacagatgta 5581ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat aatggtgcag 5641ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac cattcatgtt 5701gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt 5761gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct caacgacagg 5821agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg cttctcgccg 5881aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat 5941accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg 6001acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt 6061gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc 6121aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg 6181cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc 6241caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag 6301tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg 6361gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat 6421ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg 6481cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag 6541catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat 6601ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt 6661atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc 6721gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg 6781ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac 6841attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat 6901cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc 6961gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat 7021cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag 7081caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc 7141catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac 7201gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg 7261tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa 7321ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca 7381ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg 7441catgcaagga gatggcgccc aacagtcccc cggc SEQ ID No: 20oligonucleotide (19 b) pD-ter- 5′-AAGAAGACACGATCCACCG-3′ SEQ ID No: 21oligonucleotide (35 b) long+ 5′-CGAACGTGGCGAGAAAGGAAGGGAAGAAAGAAGTC-3′SEQ ID No: 22 oligonucleotide (71 b + 3′ modification ddC) ddC-Long25′-TTTTTTTAGACTTCTTTCTTCCCTTCCTTTCTCGCCACGTTCGAAGAAGACACGATCCACCGCCGGTTCCG-ddC-3′ SEQ ID No: 23 oligonucleotide (35 b + 3′ Biotin (TEG) Long +Tb 5′-CGAACGTGGCGAGAAAGGAAGGGAAGAAAGAAGTC-Bio-3′

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1. A reverse transcriptase comprising a Moloney Murine Leukemia Virusreverse transcriptase comprising the amino acid sequence as set forth inSEQ ID NO: 25, wherein the amino acid sequence comprises a mutation atat least position T330.
 2. The reverse transcriptase according to claim1, wherein the mutation at T330 is T330P, N, L, D, V, or S.
 3. Thereverse transcriptase according to claim 2, wherein the mutation isT330P.
 4. The reverse transcriptase of claim 1 further comprising atleast one mutation at an amino acid position chosen from: E5, M39, I49,M66, Q91, P130, L139, I179, D200, Q221, Q237, T287, A307, L333, Y344,Q374, W388, R390, Q430, D449, N479, A502, H594, L603, E607, A644, N649,D653, K658, L671, and E673,

wherein when the further mutation is at position D653 the mutation isnot D653N, and wherein when the further mutation is at position H594 themutation is not H594A.
 5. The reverse transcriptase of claim 1 furthercomprising at least one mutation at an amino acid position chosen fromL139, D200, D449, N479, or H594.
 6. The reverse transcriptase of claim5, wherein the amino acid sequence comprises a mutation at L139.
 7. Thereverse transcriptase of claim 5, wherein the amino acid sequencecomprises a mutation at D200.
 8. The reverse transcriptase of claim 5,wherein the amino acid sequence comprises a mutation at D449.
 9. Thereverse transcriptase of claim 5, wherein the amino acid sequencecomprises a mutation at N479.
 10. The reverse transcriptase of claim 5,wherein the amino acid sequence comprises a mutation at H594.
 11. Thereverse transcriptase according to claim 2, wherein the amino acidsequence comprises one of more of the following mutations: E5K, M39V orL, I49V or T, M66L, Q91R or L, P130S, L139P, I179T or V, D200N, A, or G,Q221R, Q237R, T287A, A307V, L333Q, Y344H, Q374R, W388R, R390W, Q430R,D449G or A, N479D, A502V, H594R or Q, L603W or M, E607K, G, or A, A644Vor T, N649S, D653G, A, H, or V, K658R or Q, L671P, and E673G or K.


12. The reverse transcriptase of claim 41, wherein the reversetranscriptase has increased thermostability.
 13. The reversetranscriptase of claim 1, wherein the reverse transcriptase has anoptimum activity at a temperature above 42° C.
 14. The reversetranscriptase of claim 1, wherein the reverse transcriptase has anoptimum activity at a temperature of at least 50° C.
 15. The reversetranscriptase of claim 1, wherein the reverse transcriptase has a higheractivity at 50° C. as compared with the corresponding wild type reversetranscriptase.
 16. The reverse transcriptase according to claim 1,wherein the reverse transcriptase has a specific activity at 37° C.which is at least 125% of the corresponding wild type reversetranscriptase.
 17. The reverse transcriptase of claim 1, wherein thereverse transcriptase has a thermostability of at least 1.5 times thatof the corresponding wild type reverse transcriptase, measured asresidual activity at 37° C. following treatment at 50° C. for 5 minutes.18. A polynucleotide encoding the reverse transcriptase according toclaim 1.